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Sample records for 5-aza-2 deoxycytidine treatment

  1. Increased cleavage rate of human nuclear transfer embryos after 5-aza-2'-deoxycytidine treatment.

    PubMed

    Sun, Lei; Wu, Ke-Liang; Zhang, Di; Wang, Hong-Yan; Wang, Yue; Xu, Zhen-Yu; Huang, Xiu-Ying; Chen, Zi-Jiang; Liu, Hou-Qi

    2012-10-01

    As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. The present study found that the use of cytochalasin B was not necessary for, and even harmful to, the enucleation of human zygotes. This study also decreased the DNA methylation levels in reconstructed embryos using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), in an attempt to correct the abnormalities in DNA methylation that might play an important role in the failure of embryo development. After 5-aza-dC treatment and nuclear transfer (NT-Aza group), 32.7% of reconstructed embryos developed to the 8-cell stage, which is a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that of the NT group, as determined by 5-methylcytosine immunodetection. Based on the present results, this study recommends performing the enucleation procedure without cytochalasin B treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies.

  2. 5-AZA-2'-DEOXYCYTIDINE-INDUCED DYSMORPHOGENESIS IN THE RAT

    EPA Science Inventory

    5-AZA-2'-deoxycytidine-induced dysmorphogenesis in the rat.

    Branch S, Chernoff N, Brownie C, Francis BM.

    Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695, USA. S_Branch@ncsu.edu

    5-aza-2'-deoxycytidine (d-AZA) causes tem...

  3. 5-AZA-2'-DEOXYCYTIDINE-INDUCED DYSMORPHOGENESIS IN THE RAT

    EPA Science Inventory

    5-AZA-2'-deoxycytidine-induced dysmorphogenesis in the rat.

    Branch S, Chernoff N, Brownie C, Francis BM.

    Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695, USA. S_Branch@ncsu.edu

    5-aza-2'-deoxycytidine (d-AZA) causes tem...

  4. Effect of combined 5-aza-2deoxycytidine and cisplatin treatment on the P15 lung adenocarcinoma cell line

    PubMed Central

    LIU, KAISHAN; HUANG, WENYAN; GAO, WEISONG; HE, WENFANG

    2015-01-01

    Aberrant promoter hypermethylation resulting in the epigenetic silencing of apoptosis-associated genes is a key process in the chemotherapeutic treatment of cancer. The nucleoside analog, 5-aza-2deoxycytidine (DAC), inhibits the activity of DNA methyltransferase enzymes and is able to restore the expression levels of genes that have been silenced by aberrant DNA methylation. The aim of the present study was to investigate the effect of combined treatment with DAC and cisplatin (CDDP) on the lung adenocarcinoma cell line, P15. Growth inhibition was examined using a clone formation assay and growth inhibitory activities by cell counting during treatment with DAC alone, CDDP alone or DAC followed by CDDP. In addition, changes in the mRNA expression levels of various apoptosis-associated genes following treatment with increasing concentrations of DAC were determined using reverse transcription-polymerase chain reaction. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to detect the number of apoptotic P15 tumor cells following treatment with DAC and/or CDDP. The results indicated that DAC treatment alone restored the mRNA expression levels of p73, p16INK4a, B-cell lymphoma (Bcl)-2-associated agonist of cell death and Bcl-2-associated X protein. In addition, combined therapy with DAC and CDDP was found to significantly suppress the growth of P15 tumor cells compared with DAC or CDDP treatment alone. In conclusion, DAC may enhance the chemosensitivity of the P15 cell line to treatment with CDDP. PMID:26137003

  5. Effect of combined 5-aza-2'deoxycytidine and cisplatin treatment on the P15 lung adenocarcinoma cell line.

    PubMed

    Liu, Kaishan; Huang, Wenyan; Gao, Weisong; He, Wenfang

    2015-05-01

    Aberrant promoter hypermethylation resulting in the epigenetic silencing of apoptosis-associated genes is a key process in the chemotherapeutic treatment of cancer. The nucleoside analog, 5-aza-2'deoxycytidine (DAC), inhibits the activity of DNA methyltransferase enzymes and is able to restore the expression levels of genes that have been silenced by aberrant DNA methylation. The aim of the present study was to investigate the effect of combined treatment with DAC and cisplatin (CDDP) on the lung adenocarcinoma cell line, P15. Growth inhibition was examined using a clone formation assay and growth inhibitory activities by cell counting during treatment with DAC alone, CDDP alone or DAC followed by CDDP. In addition, changes in the mRNA expression levels of various apoptosis-associated genes following treatment with increasing concentrations of DAC were determined using reverse transcription-polymerase chain reaction. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to detect the number of apoptotic P15 tumor cells following treatment with DAC and/or CDDP. The results indicated that DAC treatment alone restored the mRNA expression levels of p73, p16(INK4a) , B-cell lymphoma (Bcl)-2-associated agonist of cell death and Bcl-2-associated X protein. In addition, combined therapy with DAC and CDDP was found to significantly suppress the growth of P15 tumor cells compared with DAC or CDDP treatment alone. In conclusion, DAC may enhance the chemosensitivity of the P15 cell line to treatment with CDDP.

  6. Sequential treatment with 5-aza-2'-deoxycytidine and deacetylase inhibitors reactivates HIV-1.

    PubMed

    Bouchat, Sophie; Delacourt, Nadège; Kula, Anna; Darcis, Gilles; Van Driessche, Benoit; Corazza, Francis; Gatot, Jean-Stéphane; Melard, Adeline; Vanhulle, Caroline; Kabeya, Kabamba; Pardons, Marion; Avettand-Fenoel, Véronique; Clumeck, Nathan; De Wit, Stéphane; Rohr, Olivier; Rouzioux, Christine; Van Lint, Carine

    2015-12-17

    Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency-reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5-AzadC) in combination with clinically tolerable HDACIs in reactivating HIV-1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5-AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV-1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5-AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.

  7. Antitumor effects of a combined 5-aza-2'deoxycytidine and valproic acid treatment on rhabdomyosarcoma and medulloblastoma in Ptch mutant mice.

    PubMed

    Ecke, Ines; Petry, Frauke; Rosenberger, Albert; Tauber, Svantje; Mönkemeyer, Sven; Hess, Ina; Dullin, Christian; Kimmina, Sarah; Pirngruber, Judith; Johnsen, Steven A; Uhmann, Anja; Nitzki, Frauke; Wojnowski, Leszek; Schulz-Schaeffer, Walter; Witt, Olaf; Hahn, Heidi

    2009-02-01

    Patched (Ptch) heterozygous mice develop medulloblastoma (MB) and rhabdomyosarcoma (RMS) resembling the corresponding human tumors. We have previously shown that epigenetic silencing of the intact Ptch allele contributes to tumor formation in this model. Here, we investigated whether targeting of epigenetic silencing mechanisms could be useful in the treatment of Ptch-associated cancers. A reduction of endogenous DNA methyltransferase1 (Dnmt1) activity significantly reduced tumor incidence in heterozygous Ptch knockout mice. A combined treatment with the Dnmt inhibitor 5-aza-2'deoxycytidine (5-aza-dC) and the histone deacetlyase (HDAC) inhibitor valproic acid (VPA) efficiently prevented MB and RMS formation, whereas monotherapies with either drug were less effective. Wild-type Ptch expression was efficiently reactivated in tumors by 5-aza-dC/VPA combination therapy. This was associated with reduced methylation of the Ptch promoter and induction of histone hyperacetylation suggesting inhibition of HDACs in vivo. However, the treatment was not effective in clinically overt, advanced stage tumors. This is a first in vivo demonstration that targeting of Dnmt and HDAC activities is highly effective in preventing formation of Ptch-associated tumors. The results suggest a novel clinical strategy for consolidation therapy of corresponding tumors in humans after completion of conventional treatment. Our data also suggest that epigenetic therapy may be less effective in treating advanced stages of tumors, at least in this tumor model.

  8. 5-aza-2'-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells.

    PubMed

    Stich, Maximilian; Ganss, Lennard; Puschhof, Jens; Prigge, Elena-Sophie; Reuschenbach, Miriam; Guiterrez, Ana; Vinokurova, Svetlana; von Knebel Doeberitz, Magnus

    2016-07-16

    High-risk human papillomaviruses (hr HPVs) may cause various human cancers and associated premalignant lesions. Transformation of the host cells is triggered by overexpression of the viral oncogenes E6 and E7 that deregulate the cell cycle and induce chromosomal instability. This process is accompanied by hypermethylation of distinct CpG sites resulting in silencing of tumor suppressor genes, inhibition of the viral E2 mediated control of E6 and E7 transcription as well as deregulated expression of host cell microRNAs. Therefore, we hypothesized that treatment with demethylating agents might restore those regulatory mechanisms. Here we show that treatment with 5-aza-2'-deoxycytidine (DAC) strongly decreases the expression of E6 and E7 in a panel of HPV-transformed cervical cancer and head and neck squamous cell carcinoma cell lines. Reduction of E6 and E7 further resulted in increased target protein levels including p53 and p21 reducing the proliferation rates and colony formation abilities of the treated cell lines. Moreover, DAC treatment led to enhanced expression of tumor the suppressive miRNA-375 that targets and degrades E6 and E7 transcripts. Therefore, we suggest that DAC treatment of HPV-associated cancers and respective precursor lesions may constitute a targeted approach to subvert HPV oncogene functions that deserves testing in clinical trials.

  9. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing

    PubMed Central

    Klar, Agnes S.; Gopinadh, Jakka; Kleber, Sascha; Wadle, Andreas; Renner, Christoph

    2015-01-01

    Background NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC). Methodology/Principal Findings We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157–165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels. Conclusions/Significance These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment. PMID:26447882

  10. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    PubMed

    Klar, Agnes S; Gopinadh, Jakka; Kleber, Sascha; Wadle, Andreas; Renner, Christoph

    2015-01-01

    NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC). We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels. These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  11. [Effect of 5-aza-2'-deoxycytidine on DAPK gene expression in human HL-60 cells].

    PubMed

    Wang, Chun-Yan; Liu, Wen-Jun

    2014-06-01

    This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.

  12. 5-AZA-2'-DEOXYCYTIDINE INDUCED CYTOTOXICITY AND LONG BONE REDUCTION DEFECTS IN THE MURINE LIMB

    EPA Science Inventory

    The antineoplastic drug 5-aza-2'-deoxycytidine (dAZA) is a DNA hypomethylating agent that can be used to induce hind limb phocomelia in the offspring of CD-1 Swiss Webster mice. Previously, our laboratory investigated the possibility that dAZA induced alterations in gene express...

  13. 5-AZA-2'-DEOXYCYTIDINE INDUCED CYTOTOXICITY AND LONG BONE REDUCTION DEFECTS IN THE MURINE LIMB

    EPA Science Inventory

    The antineoplastic drug 5-aza-2'-deoxycytidine (dAZA) is a DNA hypomethylating agent that can be used to induce hind limb phocomelia in the offspring of CD-1 Swiss Webster mice. Previously, our laboratory investigated the possibility that dAZA induced alterations in gene express...

  14. Hypomethylating agent 5-aza-2'-deoxycytidine (DAC) ameliorates multiple sclerosis in mouse models.

    PubMed

    Mangano, Katia; Fagone, Paolo; Bendtzen, Klaus; Meroni, Pier Luigi; Quattrocchi, Cinzia; Mammana, Santa; Di Rosa, Michelino; Malaguarnera, Lucia; Coco, Marinella; Magro, Gaetano; Di Marco, Roberto; Nicoletti, Ferdinando

    2014-12-01

    Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) in two murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease was associated with a reduction in the number of spinal cord infiltrating lymphocytes and amelioration of the histopathological signs associated with EAE. In addition, increased transcript levels of anti-inflammatory cytokines and decreased mRNA expression of pro-inflammatory mediators were also observed. Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3.

  15. Human Leukocyte Antigen-G Is Frequently Expressed in Glioblastoma and May Be Induced in Vitro by Combined 5-Aza-2′-Deoxycytidine and Interferon-γ Treatments

    PubMed Central

    Wastowski, Isabela J.; Simões, Renata T.; Yaghi, Layale; Donadi, Eduardo A.; Pancoto, João T.; Poras, Isabelle; Lechapt-Zalcman, Emmanuèle; Bernaudin, Myriam; Valable, Samuel; Carlotti, Carlos G.; Flajollet, Sébastien; Jensen, Stine S.; Ferrone, Soldano; Carosella, Edgardo D.; Kristensen, Bjarne W.; Moreau, Philippe

    2014-01-01

    Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2′-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2′-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G–negative tumors. PMID:23219427

  16. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells

    PubMed Central

    Vispé, Stéphane; Deroide, Arthur; Davoine, Emeline; Desjobert, Cécile; Lestienne, Fabrice; Fournier, Lucie; Novosad, Natacha; Bréand, Sophie; Besse, Jérôme; Busato, Florence; Tost, Jörg; De Vries, Luc; Cussac, Didier; Riond, Joëlle; Arimondo, Paola B.

    2015-01-01

    5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia. PMID:25948775

  17. Vitamin C increases viral mimicry induced by 5-aza-2′-deoxycytidine

    PubMed Central

    Liu, Minmin; Ohtani, Hitoshi; Zhou, Wanding; Ørskov, Andreas Due; Charlet, Jessica; Zhang, Yang W.; Shen, Hui; Baylin, Stephen B.; Liang, Gangning; Grønbæk, Kirsten; Jones, Peter A.

    2016-01-01

    Vitamin C deficiency is found in patients with cancer and might complicate various therapy paradigms. Here we show how this deficiency may influence the use of DNA methyltransferase inhibitors (DNMTis) for treatment of hematological neoplasias. In vitro, when vitamin C is added at physiological levels to low doses of the DNMTi 5-aza-2′-deoxycytidine (5-aza-CdR), there is a synergistic inhibition of cancer-cell proliferation and increased apoptosis. These effects are associated with enhanced immune signals including increased expression of bidirectionally transcribed endogenous retrovirus (ERV) transcripts, increased cytosolic dsRNA, and activation of an IFN-inducing cellular response. This synergistic effect is likely the result of both passive DNA demethylation by DNMTi and active conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten–eleven translocation (TET) enzymes at LTR regions of ERVs, because vitamin C acts as a cofactor for TET proteins. In addition, TET2 knockout reduces the synergy between the two compounds. Furthermore, we show that many patients with hematological neoplasia are markedly vitamin C deficient. Thus, our data suggest that correction of vitamin C deficiency in patients with hematological and other cancers may improve responses to epigenetic therapy with DNMTis. PMID:27573823

  18. [5-Aza-2'-deoxycytidine enhances differentiation and apoptosis induced by phenylbutyrate in Kasumi-1 cells].

    PubMed

    Hao, Chang-lai; Tang, Ke-jing; Chen, Sen; Xing, Hai-yan; Wang, Min; Wang, Jian-xiang

    2005-03-01

    To investigate whether phenylbutyrate (PB) combined with 5-aza-2'-deoxycytidine (5-Aza-CdR)could inhibit transcription repression and induce t(8;21) acute myelogenous leukemia (AML) Kasumi-1 cells to differentiate and undergo apoptosis. Kasumi-1 cells were treated with PB and 5-Aza-CdR at different concentrations in suspension culture. Cellular proliferation was determined by the MTT assay, expression of myeloid-specific differentiation antigen and cell cycles were analyzed by flow cytometry. Cell apoptosis were assessed using AnnexinV/PI staining and flow cytometry. Treatment of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC(50) of 2.3 mmol/L. When combined with 5-Aza-CdR, PB resulted in a greater growth inhibition with an IC(50) of 1.95 mmol/L. Treatment of Kasumi-1 cells with PB resulted in cell cycle arrest at G(0)/G(1), while combined treatment with PB and 5-Aza-CdR led to cell cycle arrest at G(2)/M. Expression of myeloid cell differentiation antigens CD11b and CD13 induced by PB was enhanced when Kasumi-1 cells were pretreated with low dose of 5-Aza-CdR. High, but not low, concentrations of 5-Aza-CdR could enhance early apoptosis of Kasumi-1 cells induced by PB. Phenylbuty rate, when combined with 5-Aza-CdR, inhibits AML cell in vitro proliferation and increases apoptosis in a synergistic fashion.

  19. DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

    PubMed Central

    Flatau, E; Gonzales, F A; Michalowsky, L A; Jones, P A

    1984-01-01

    A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines. PMID:6209556

  20. Non-intensive treatment with low-dose 5-aza-2'-deoxycytidine (DAC) prior to allogeneic blood SCT of older MDS/AML patients.

    PubMed

    Lübbert, M; Bertz, H; Rüter, B; Marks, R; Claus, R; Wäsch, R; Finke, J

    2009-11-01

    Novel, non-intensive treatment options in older MDS/AML patients planned for allografting, with the goal of down-staging the underlying disease and bridging time to transplantation, are presently being developed. 5-azacytidine and decitabine (DAC) are of particular interest, as they can be given repetitively, with very limited non-hematologic toxicity and result in responses both in MDS and AML even at low doses. We describe 15 consecutive patients (median age 69 years, range 60-75 years) with MDS (n=10) or AML (n=5) who all received first-line treatment with DAC and subsequent allografting (from sibling donor in four patients, unrelated donor in 11) after reduced-intensity conditioning with the FBM regimen. Successful engraftment was attained in 14/15 patients, all of whom achieved a CR, with a median duration of 5 months (range 1+ to 51+). Six of these 14 patients are alive (4 with complete donor chimerism), 8 have died either from relapse (n=4) or treatment-related complications while in CR (n=4). We conclude that allografting after low-dose DAC and subsequent conditioning with FBM is feasible, with no unexpected toxicities and appears as a valid alternative to standard chemotherapy ('InDACtion instead of induction') in elderly patients with MDS/AML.

  1. Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.

    PubMed

    Saini, M; Selokar, N L; Agrawal, H; Singla, S K; Chauhan, M S; Manik, R S; Palta, P

    2016-04-01

    We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

  2. DNA demethylation caused by 5-Aza-2′-deoxycytidine induces mitotic alterations and aneuploidy

    PubMed Central

    Lentini, Laura; Cilluffo, Danilo; Di Leonardo, Aldo

    2016-01-01

    Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation. Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects. Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore. PMID:26771138

  3. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells.

    PubMed

    Zych, J; Stimamiglio, M A; Senegaglia, A C; Brofman, P R S; Dallagiovanna, B; Goldenberg, S; Correa, A

    2013-05-01

    Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

  4. Effects of 5-aza-2deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

    PubMed Central

    Zhou, X.Q.; Huang, S.Y.; Zhang, D.S.; Zhang, S.Z.; Li, W.G.; Chen, Z.W.; Wu, H.W.

    2014-01-01

    Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment. PMID:25517920

  5. Effects of 5-Aza-2'-Deoxycytidine, Bromodeoxyuridine, Interferons and Hydrogen Peroxide on Cellular Senescence in Cholangiocarcinoma Cells.

    PubMed

    Moolmuang, Benchamart; Singhirunnusorn, Pattama; Ruchirawat, Mathuros

    2016-01-01

    Cellular senescence, a barrier to tumorigenesis, controls aberrant proliferation of cells. We here aimed to investigate cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines using five different inducing agents: 5-aza-2'deoxycytidine, bromodeoxyuridine, interferons (IFNβ and IFNγ), and hydrogen peroxide. We analyzed senescence characteristics, colony formation ability, expression of genes involved in cell cycling and interferon signaling pathways, and protein levels. Treatment with all five agents decreased cell proliferation and induced cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines with different degrees of growth-inhibitory effects depending on cell type and origin. Bromodeoxyuridine gave the strongest stimulus to inhibit growth and induce senescence in most cell lines tested. Expression of p21 and interferon related genes was upregulated in most conditions. The fact that bromodeoxyuridine had the strongest effects on growth inhibition and senescence induction implies that senescence in cholangiocarcinoma cells is likely controlled by DNA damage response pathways relating to the p53/p21 signaling. In addition, interferon signaling pathways may partly regulate this mechanism in cholangiocarcinoma cells.

  6. Synergy of 5-aza-2'-deoxycytidine (DAC) and paclitaxel in both androgen-dependent and -independent prostate cancer cell lines.

    PubMed

    Shang, Donghao; Liu, Yuting; Liu, Qingjun; Zhang, Fengbo; Feng, Lang; Lv, Wencheng; Tian, Ye

    2009-06-08

    To determine the synergy of 5-aza-2'-deoxycytidine (DAC) and paclitaxel (PTX) against prostate carcinoma (PC) cells by isobolographic analysis. We demonstrated that DAC could significantly increase the susceptibility of PC cells to PTX, and confirmed the synergy of DAC and PTX. DAC enhanced the PTX induced up-regulation of caspase activity and antiproliferative effect, resulting in an increase of cells in subG1 and G2/M phases. In addition, the synergy was observed in both androgen-dependent and -independent PC cell lines. It suggested that combination chemotherapy with DAC and PTX might be a new strategy to improve the clinical response rate of PC.

  7. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

    PubMed Central

    Zych, J.; Stimamiglio, M.A.; Senegaglia, A.C.; Brofman, P.R.S.; Dallagiovanna, B.; Goldenberg, S.; Correa, A.

    2013-01-01

    Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available. PMID:23797495

  8. Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine.

    PubMed

    Jafarpour, Farnoosh; Hosseini, Sayed Morteza; Hajian, Mahdi; Forouzanfar, Mohsen; Abedi, Parvaneh; Hosseini, Laleh; Ostadhosseini, Somaye; Gholami, Soghra; Nasr Esfahani, Mohammad Hossein

    2011-01-01

    Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 μM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4. In comparison with untreated cells, 0.01 and 0.08 μM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 μM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 μM 5-aza-dc treated cells when compared with 0 and 0.01 μM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 μM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 μM 5-aza-dc remained unchanged. These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.

  9. 5,6-Dihydro-5-aza-2'-deoxycytidine potentiates the anti-HIV-1 activity of ribonucleotide reductase inhibitors.

    PubMed

    Rawson, Jonathan M; Heineman, Richard H; Beach, Lauren B; Martin, Jessica L; Schnettler, Erica K; Dapp, Michael J; Patterson, Steven E; Mansky, Louis M

    2013-11-15

    The nucleoside analog 5,6-dihydro-5-aza-2'-deoxycytidine (KP-1212) has been investigated as a first-in-class lethal mutagen of human immunodeficiency virus type-1 (HIV-1). Since a prodrug monotherapy did not reduce viral loads in Phase II clinical trials, we tested if ribonucleotide reductase inhibitors (RNRIs) combined with KP-1212 would improve antiviral activity. KP-1212 potentiated the activity of gemcitabine and resveratrol and simultaneously increased the viral mutant frequency. G-to-C mutations predominated with the KP-1212-resveratrol combination. These observations represent the first demonstration of a mild anti-HIV-1 mutagen potentiating the antiretroviral activity of RNRIs and encourage the clinical translation of enhanced viral mutagenesis in treating HIV-1 infection. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. [Combination of phenylbutyrate and 5-Aza-2'deoxycytidine inhibits human Kasumi-1 xenograft tumor growth in nude mice].

    PubMed

    Hao, Chang-lai; Lin, Dong; Wang, Li-hong; Xing, Hai-yan; Wang, Min; Wang, Jian-Xiang

    2004-11-01

    To investigate the tumor suppression efficacy of histone deacetylase inhibitor, phenylbutyrate (PB), in combination with DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza-CdR) in the treatment of Kasumi-1 xenograft tumor in nude mice and its mechanism. The nude mice model of Kasumi-1 xenograft tumor was established by subcutaneous inoculation. Latency of tumor formation, the ability of Kasumi-1 cells pre treated with PB to form the xenograft tumor, and the tumor suppression activity of PB and 5-Aza-CdR by intraperitoneal injection in xenografted mice model were detected. Cell differentiation and cell cycle parameters of the tumor cells were analyzed by flow cytometry analysis, apoptosis by TUNEL in situ hybridization, and tumor microvessel density (MVD) by immunohistochemistry study. The latency of tumor formation in mice with or without previous lienectomy was 17 approximately 23 and 40 approximately 50 days, respectively. Tumor cells xenografted could not be found in other tissues than in inoculation area, and still harbored the specific t(8;21) and AML1-ETO fusion gene. When the xenografted mice models treated with PB, 5-Aza-CdR, or both, the tumor growth inhibition rates were 49.07%, 25.69% and 87.46% (P < 0.05), the apoptosis indexes (AI) of tumor cells were (2.25 +/- 0.85)%, (1.32 +/- 0.68)%, and (5.41 +/- 1.56)% (P < 0.05), and the microvessel densities (MVD) were 21.69 +/- 6.25, 28.34 +/- 4.24 and 9.48 +/- 3.21 (P < 0.01), respectively. All the data above were significantly different from that in control (P < 0.05). The expression of CD11b and CD13 antigen of the tumor cells was increased in xenografted mice model treated with PB when compared with the control \\[(12.08 +/- 1.02)% and (54.91 +/- 2.72)%\\], respectively (P < 0.01), and tumor cells showed a cell cycle arrest with increased G(0)/G(1)-phase cells and decreased S-phase cells. PB inhibited the growth of Kasumi-1 xenograft tumor by inducing tumor cell apoptosis and differentiation, and

  11. Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

    PubMed

    Huan, Yan Jun; Zhu, Jiang; Xie, Bing Teng; Wang, Jian Yu; Liu, Shi Chao; Zhou, Yang; Kong, Qing Ran; He, Hong Bin; Liu, Zhong Hua

    2013-10-01

    The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

  12. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

    PubMed Central

    2016-01-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  13. Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'- deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT

    PubMed Central

    1992-01-01

    The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells. PMID:1371288

  14. Identification of a class of human cancer germline genes with transcriptional silencing refractory to the hypomethylating drug 5-aza-2′-deoxycytidine.

    PubMed Central

    Almatrafi, Ahmed; Feichtinger, Julia; Vernon, Ellen G.; Escobar, Natalia Gomez; Wakeman, Jane A.; Larcombe, Lee D.; McFarlane, Ramsay J.

    2014-01-01

    Bona fide germline genes have expression restricted to the germ cells of the gonads. Testis-specific germline development-associated genes can become activated in cancer cells and can potentially drive the oncogenic process and serve as therapeutic/biomarker targets; such germline genes are referred to as cancer/testis genes. Many cancer/testis genes are silenced via hypermethylation of CpG islands in their associated transcriptional control regions and become activated upon treatment with DNA hypomethylating agents; such hypomethylation-induced activation of cancer/testis genes provides a potential combination approach to augment immunotherapeutics. Thus, understanding cancer/testis gene regulation is of increasing clinical importance. Previously studied cancer/testis gene activation has focused on X chromosome encoded cancer/testis genes. Here we find that a sub-set of non-X encoded cancer/testis genes are silenced in non-germline cells via a mechanism that is refractory to epigenetic dysregulation, including treatment with the hypomethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor tricostatin A. These findings formally indicate that there is a sub-group of the clinically important cancer/testis genes that are unlikely to be activated in clinical therapeutic approaches using hypomethylating agents and it indicates a unique transcriptional silencing mechanism for germline genes in non-germline cells that might provide a target mechanism for new clinical therapies. PMID:25594001

  15. Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.

    PubMed

    Choi, Ji-Young; Lee, Sangmi; Hwang, Soojin; Jo, Sangmee Ahn; Kim, Miji; Kim, Young Ju; Pang, Myung-Geol; Jo, Inho

    2013-01-01

    5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.

  16. The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2deoxycytidine (decitabine) are differentially regulated by cell cycle progression

    PubMed Central

    Yu, Margaret; Burnett, David M; Alexander, Amanda; Samlowski, Wolfram; Fitzpatrick, Frank A

    2011-01-01

    5-Aza-2′-deoxycytidine (decitabine) is a drug targeting the epigenetic abnormalities of tumors. The basis for its limited efficacy in solid tumors is unresolved, but may relate to their indolent growth, their p53 genotype or both. We report that the primary molecular mechanism of decitabine—depletion of DNA methyltransferase-1 following its “suicide” inactivation—is not absolutely associated with cell cycle progression in HCT 116 colon cancer cells, but is associated with their p53 genotype. Control experiments affirmed that the secondary molecular effects of decitabine on global and promoter-specific CpG methylation and MAGE-A1 mRNA expression were S-phase dependent, as expected. Secondary changes in CpG methylation occurred only in growing cells ∼24–48 h after decitabine treatment; these epigenetic changes coincided with p53 accumulation, an index of DNA damage. Conversely, primary depletion of DNA methyltransferase-1 began immediately after a single exposure to 300 nM decitabine and it progressed to completion within ∼8 h, even in confluent cells arrested in G1 and G2/M. Our results suggest that DNA repair and remodeling activity in arrested, confluent cells may be sufficient to support the primary molecular action of decitabine, while its secondary, epigenetic effects require cell cycle progression through S-phase. PMID:21725200

  17. Simultaneous quantitative determination of 5-aza-2'-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors.

    PubMed

    Anders, Nicole M; Liu, Jianyong; Wanjiku, Teresia; Giovinazzo, Hugh; Zhou, Jianya; Vaghasia, Ajay; Nelson, William G; Yegnasubramanian, Srinivasan; Rudek, Michelle A

    2016-06-01

    The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.

  18. 5-Aza-2'-deoxycytidine suppresses human renal carcinoma cell growth in a xenograft model via up-regulation of the connexin 32 gene.

    PubMed

    Hagiwara, H; Sato, H; Ohde, Y; Takano, Y; Seki, T; Ariga, T; Hokaiwado, N; Asamoto, M; Shirai, T; Nagashima, Y; Yano, T

    2008-04-01

    The connexin (Cx) 32 gene, a member of the gap junction gene family, acts as a tumour suppressor gene in human renal cell carcinoma (RCC) and is down-regulated by the hypermethylation of CpG islands in a promoter region of the Cx gene. The current study investigated whether the restoration of Cx32 silenced by hypermethylation in RCC by a DNA demethylating agent could be an effective treatment against RCC. Using nude mice bearing Caki-1 cells (a human metastatic RCC cell line), the effects of 5-aza-2'-deoxycytidine (5-aza-CdR), a DNA demethylase inhibitor, on Cx32 mRNA expression and tumour growth were examined by RT-PCR, and by measuring tumour weight and volume. Cx32 expression in Caki-1 tumours was inhibited by Cx32 short interfering (si) RNA, and the effect of siRNA on 5-aza-CdR-dependent suppression of tumour growth in nude mice was evaluated. 5-aza-CdR treatment inhibited the growth of Caki-1 cells in nude mice by 70% and increased 7-fold the level of Cx32 mRNA. The intratumour injection of Cx32 siRNA almost totally inhibited the expression of Cx32 mRNA and significantly reduced the suppression of tumour growth in 5-aza-CdR-treated nude mice. 5-aza-CdR suppressed the growth of Caki-1 tumours in a xenograft model, by restoring Cx32 expression. This finding suggests that treatment with 5-aza-CdR could be a new effective therapy against human metastatic RCC and that Cx32 could be a potential target for the treatment of RCC.

  19. Epigenetic modulation of AR gene expression in prostate cancer DU145 cells with the combination of sodium butyrate and 5'-Aza-2'-deoxycytidine.

    PubMed

    Fialova, Barbora; Luzna, Petra; Gursky, Jan; Langova, Katerina; Kolar, Zdenek; Trtkova, Katerina Smesny

    2016-10-01

    The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. Castration-resistant prostate cancer (CRPC) is a consequence of androgen deprivation therapy. Unchecked CRPC followed by metastasis is lethal. Some CRPCs show decreased AR gene expression due to epigenetic mechanisms such as DNA methylation and histone deacetylation. The aim of this study was to epigenetically modulate the methylated state of the AR gene leading to targeted demethylation and AR gene expression in androgen-independent human prostate cancer DU145 cell line, representing the CRPC model with very low or undetectable AR levels. The cell treatment was based on single and combined applications of two epigenetic inhibitors, sodium butyrate (NaB) as histone deacetylases inhibitor and 5'-Aza-2'-deoxycytidine (Aza-dC) as DNA methyltransferases inhibitor. We found that the Aza-dC in combination with NaB may help reduce the toxicity of higher NaB concentrations in cancer cells. In normal RWPE-1 cells and even stronger in cancer DU145 cells, the combined treatment induced both AR gene expression on the mRNA level and increased histone H4 acetylation in AR gene promoter. Also activation and maintenance of G2/M cell cycle arrest and better survival in normal RWPE-1 cells compared to cancer DU145 cells were observed after the treatments. These results imply the selective toxicity effect of both inhibitors used and their potentially more effective combined use in the epigenetic therapy of prostate cancer patients.

  20. The DNA hypomethylating agent, 5-aza-2'-deoxycytidine, enhances tumor cell invasion through a transcription-dependent modulation of MMP-1 expression in human fibrosarcoma cells.

    PubMed

    Poplineau, Mathilde; Schnekenburger, Michael; Dufer, Jean; Kosciarz, Aleksandra; Brassart-Pasco, Sylvie; Antonicelli, Frank; Diederich, Marc; Trussardi-Régnier, Aurélie

    2015-01-01

    In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5-aza-2'-deoxycytidine (5-azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP-1, -2, and -9 in human HT1080 fibrosarcoma cells. We demonstrate that 5-azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad-spectrum and specific MMP inhibitors, we establish that MMP-1, but not MMP-2 and -9, plays a key role in 5-azadC-enhanced cell invasion. We show that 5-azadC induces MMP-1 expression through a transcriptional mechanism without affecting MMP-1 promoter methylation status. Finally, we demonstrate that 5-azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP-1 promoter, resulting in chromatin remodeling associated to 5-azadC-induced MMP-1 expression. All together, our data indicate that the hypomethylating agent 5-azadC modulates, mainly via Sp1 recruitment, MMP-1 expression resulting in an increased invasive potential of HT1080 cells. © 2013 Wiley Periodicals, Inc.

  1. A Systematic Assessment of Radiation Dose Enhancement by 5-Aza-2'-Deoxycytidine and Histone Deacetylase Inhibitors in Head-and-Neck Squamous Cell Carcinoma

    SciTech Connect

    Schutter, Harlinde de; Kimpe, Marlies; Isebaert, Sofie; Nuyts, Sandra

    2009-03-01

    Purpose: Investigations of epigenetic drugs have shown that radiotherapy can be successfully combined with histone deacetylase inhibitors (HDAC-Is) for the treatment of head-and-neck squamous cell carcinoma (HNSCC). Whether the reversal of epigenetic silencing by demethylating agents with or without HDAC-Is can also act as radiosensitizing remains unclear. This study therefore aimed to investigate whether 5-aza-2'-deoxycytidine (DAC) alone or in combination with the HDAC-Is trichostatin A, LBH589, or MGCD0103 could radiosensitize HNSCC tumor cell lines. Methods and Materials: Histone acetylation status and expression of epigenetically silenced genes at the DNA, RNA, and protein levels were assessed as measures of drug effectiveness in six HNSCC cell lines. Based on their colony-forming capacity, colony assays were performed in four of six cell lines to evaluate the radiosensitizing potential of DAC with or without HDAC-Is. Additional assays of cell survival, apoptosis, cell proliferation, and DNA damage were performed. Results: Radiosensitization was observed in two HNSCC cell lines treated with noncytotoxic doses of DAC with or without HDAC-Is before irradiation. The radiosensitizing doses induced histone hyperacetylation and reversal of gene silencing to variable extents and increased radiation-induced cell-cycle arrest. Conclusions: A role for low-dose DAC with or without HDAC-Is as radiosensitizers in HNSCC seems promising and is supportive of future clinical use, especially for combinations of DAC with LBH589 or MGCD0103, although the mechanisms by which they work will require further study.

  2. Role of spermatogonial stem cells extract in transdifferentiation of 5-Aza-2'-deoxycytidine-treated bone marrow mesenchymal stem cells into germ-like cells.

    PubMed

    Kharizinejad, Ebrahim; Minaee Zanganeh, Bagher; Khanlarkhani, Neda; Mortezaee, Keywan; Rastegar, Tayebeh; Baazm, Maryam; Abolhassani, Farid; Sajjadi, Seyed Mehdi; Hajian, Mahdieh; Aliakbari, Fereshte; Barbarestani, Mohammad

    2016-05-01

    As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSCS ) extract is considered as new approach in stem cell therapy of infertility. 5-aza-2'-deoxycytidine (5-aza-dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSCS extract incubation in 5-aza-dC-treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5-aza-dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSCS extract; BMMSCs were then incubated with SSCS extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5-aza-dC and extract-5-aza-dC. After one week of incubation, flow cytometry and real-time polymerase chain reaction (PCR) exhibited high levels of expression for β1- and α6-integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract-5-aza-dC groups (P < 0.05 vs. control and 5-aza-dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ-like cells. 5-aza-dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ-like cells; this strategy could introduce a new approach for treatment of male infertility in clinic.

  3. [Effects of 5-Aza-2'-deoxycytidine on the carcinogenesis of colorectal cancer in mouse and the in vivo expression of p16/CDKN(2) mRNA].

    PubMed

    Fang, Xiao-Ming; Jiang, Zhao-Hui; Yao, Ning; Ding, Xiao-Wen; Peng, Jia-Ping; Zheng, Shu

    2011-09-06

    To explore the effects and relationship of specific demethylation agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on colorectal cancer (CRC) induced by 1, 2-dimethylhydrazine (DMH) in mouse and the in vivo expression of cyclin-dependent kinases inhibitor p16/CDKN(2) mRNA. A total of 40 male KM mice were randomized into 2 groups and CRC was induced by a 22-week injection of DMH. One group was interfered by specific DNA methyltransferase inhibitor 5-Aza-CdR. Another 10 the same source male KM mice were induced by a 22-week injection of saline as none induced cancer control group (negative control group). All mice were sacrificed to examine for colorectal neoplasm. Immunohistochemical staining was used to assess the expression of proliferating cell nuclear antigen (PCNA). The expression of p16/CDKN(2) mRNA was detected by in situ hybridization. The average numbers of neoplasm was higher in the DMH group (7.6 ± 3.1) than that of the group DMH + 5-Aza-CdR (3.4 ± 1.8, P < 0.05). Immunohistochemical staining showed there was a significant elevation of PCNA in the group DMH (16/19) as compared with that in the group DMH + 5-Aza-CdR (11/19, P < 0.05). In situ hybridization revealed that the level of tumor suppressor gene p16/CDKN(2) mRNA was significantly lower in the group DMH than that in the group DMH + 5-Aza-CdR. The specific demethylation agent 5-Aza-2'-deoxycytidine may inhibit the carcinogenesis of CRC. Its mechanism may be related with a high expression of p16/CDKN(2) mRNA.

  4. Generation of antigen-presenting cells from tumor-infiltrated CD11b myeloid cells with DNA demethylating agent 5-aza-2'-deoxycytidine.

    PubMed

    Daurkin, Irina; Eruslanov, Evgeniy; Vieweg, Johannes; Kusmartsev, Sergei

    2010-05-01

    Tumor-recruited CD11b myeloid cells, including myeloid-derived suppressor cells, play a significant role in tumor progression, as these cells are involved in tumor-induced immune suppression and tumor neovasculogenesis. On the other hand, the tumor-infiltrated CD11b myeloid cells could potentially be a source of immunostimulatory antigen-presenting cells (APCs), since most of these cells represent common precursors of both dendritic cells and macrophages. Here, we investigated the possibility of generating mature APCs from tumor-infiltrated CD11b myeloid cells. We demonstrate that in vitro exposure of freshly excised mouse tumors to DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (decitabine, AZA) results in selective elimination of tumor cells, but, surprisingly it also enriches CD45(+) tumor-infiltrated cells. The majority of "post-AZA" surviving CD45(+) tumor-infiltrated cells were represented by CD11b myeloid cells. A culture of isolated tumor-infiltrated CD11b cells in the presence of AZA and GM-CSF promoted their differentiation into mature F4/80/CD11c/MHC class II-positive APCs. These tumor-derived myeloid APCs produced substantially reduced amounts of immunosuppressive (IL-13, IL-10, PGE(2)), pro-angiogenic (VEGF, MMP-9) and pro-inflammatory (IL-1beta, IL-6, MIP-2) mediators than their precursors, freshly isolated tumor-infiltrated CD11b cells. Vaccinating naïve mice with ex vivo generated tumor-derived APCs resulted in the protection of 70% mice from tumor outgrowth. Importantly, no loading of tumor-derived APC with exogenous antigen was needed to stimulate T cell response and induce the anti-tumor effect. Collectively, our results for the first time demonstrate that tumor-infiltrated CD11b myeloid cells can be enriched and differentiated in the presence of DNA demethylating agent 5-aza-2'-deoxycytidine into mature tumor-derived APCs, which could be used for cancer immunotherapy.

  5. [Effects of 5-Aza-2'-deoxycytidine and trichostatin A on P16, hMLH1 and MGMT genes and DNA methylation in human gastric cancer cells].

    PubMed

    Meng, Chun-feng; Zhu, Xin-jiang; Dai, Dong-qiu; Peng, Guo

    2009-09-01

    To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) on DNA methylation and expression of P16, hMLH1 and MGMT genes in the human gastric cancer cell line MGC-803, and to explore the mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. MGC-803 cells were cultured in RPMI-1640 medium and were treated with 5-Aza-dC or TSA. Methylation-specific polymerase chain reaction (MS-PCR) was used to detect the promoter methylation status of P16, hMLH1 and MGMT genes. RT-PCR was used to detect the mRNA expressions of P16, hMLH1 and MGMT. Promoter hypermethylation of P16, hMLH1 and MGMT genes were detected in MGC-803 cells, and mRNA expressions of P16, hMLH1 and MGMT were absent before treatment. After treatment with 5-Aza-dC, the promoter region of the P16, hMLH1 and MGMT gene exhibited a demethylation status, and their mRNA expressions were increased. The treatment with TSA had no effects on DNA demethylation or restoration of P16 or hMLH1 expression. P16, hMLH1 and MGMT mRNA relative expression levels after treatment with a combination of 5-Aza-dC and TSA were 0.412+/-0.030, 0.397+/-0.024 and 0.553+/-0.043 respectively, which were higher than those after 5-Aza-dC treatment alone (0.221+/-0.022, 0.214+/-0.018 and 0.156+/-0.017, all P<0.05). Promoter hypermethylation is a major mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. Treatment with 5-Aza-dC alone or the combination of 5-Aza-dC and TSA can reactivate the expressions of these genes.

  6. Simultaneous quantitative determination of 5-aza-2′-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

    PubMed Central

    Anders, Nicole M.; Liu, Jianyong; Wanjiku, Teresia; Giovinazzo, Hugh; Zhou, Jianya; Vaghasia, Ajay; Nelson, William G.; Yegnasubramanian, Srinivasan; Rudek, Michelle A.

    2016-01-01

    The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2′-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2′-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2′-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100 mm × 2.1 mm, 5μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 minute total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2′-deoxycytidine, 2′-deoxycytidine, and 5-methyl-2′-deoxycytidine. The assay range was 2 – 400 ng/mL for 5-aza-2′-deoxycytidine, 50 – 10,000 ng/mL for 2′-deoxycytidine, and was 5 – 1,000 ng/mL for 5-methyl-2′-deoxycytidine. The assay proved to be accurate (93.0–102.2%) and precise (CV ≤ 6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at −70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2′-deoxycytidine and 5-methyl-2′-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine. PMID:27082761

  7. Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

    PubMed Central

    HUAN, Yan Jun; ZHU, Jiang; XIE, Bing Teng; WANG, Jian Yu; LIU, Shi Chao; ZHOU, Yang; KONG, Qing Ran; HE, Hong Bin; LIU, Zhong Hua

    2013-01-01

    The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression. PMID

  8. 5-Aza-2′-deoxycytidine Sensitizes Busulfan-resistant Myeloid Leukemia Cells By Regulating Expression of Genes Involved in Cell Cycle Checkpoint and Apoptosis

    PubMed Central

    Valdez, Benigno C.; Li, Yang; Murray, David; Corn, Paul; Champlin, Richard E.; Andersson, Borje S.

    2009-01-01

    Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2′-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu2506 ML cells to 0.5 μM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16INK4A, activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05 μM DAC and 5 μg/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu2506 cells using pifithrin-α alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell-cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pre-transplant regimen in patients with ML. PMID:19732952

  9. [Effect of arsenic trioxide and 5-aza-2'-deoxycytidine on SHP-1, JAK3, TYK2 gene expression in K562 cells].

    PubMed

    Zhang, Xiao-Kun; Luo, Jian-Min; Sun, Jie

    2014-04-01

    This study was purposed to explore the effects of a methylation inhibitor arsenic trioxide (As2O3, ATO) and 5-Aza-2'-deoxycytidine (5-aza-CdR) on the expression of JAK-STAT signal transduction pathway in family members JAK3, TYK2 and hematopoietic cell phosphatase SHP-1 in chronic myeloid leukemia cell line K562 and their roles in pathogenesis of leukemia. The K562 cells were divided into 3 groups:single drug-treated group, combined 2 drugs-treated group, group without drug treatment as control. The concentration of 5-aza-CdR were 0.5, 1, 2 µmol/L; the concentration of ATO was 1, 2.5, 5 µmol/L; the concentration of combined drugs was ATO 1 µmol/L + 5-aza-CdR 0.5 µmol/L, ATO 2.5 µmol/L + 5-aza-CdR 1 µmol/L, and ATO 5 µmol/L + 5-aza-CdR 2 µmol/L. The K562 cells were treated with above-mentioned concentration of drugs for 24, 48 and 72 hours, then the total RNA of cells was extracted, the JAK3, TYK2 and SHP-1 expressions were detected by real-time quantitative-PCR. The results showed that after the K562 cells were treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 mRNA increased, the expressions of JAK3 mRNA and TYK2 mRNA decreased along with increasing of concentration and prolonging of time, displaying the concentration and time-dependency. The SHP-1 negatively related with JAK3 and TYK2. The effect of SHP-1 on JAK3 was significantly higher than that on TYK2. It is concluded that when the K562 cells are treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 is up-regulated and the expressions of JAK3, TYK2 are down-regulated in concentration-and time-dependent manners, moreover the ATO and 5-aza-CdR show synergies demethylation effect. The SHP-1 gene exert effect possibly through inhibiting the JAK/STAT pathway, the JAK3 is affected more than TYK2, the JAK3 may exert more important role in TAK/STAT pathway.

  10. Transcriptional reactivation of murine cytomegalovirus ie gene expression by 5-aza-2'-deoxycytidine and trichostatin A in latently infected cells despite lack of methylation of the major immediate-early promoter.

    PubMed

    Hummel, Mary; Yan, Shixian; Li, Zhigao; Varghese, Thomas K; Abecassis, Michael

    2007-04-01

    We have used a spleen explant model to investigate mechanisms of murine cytomegalovirus latency and reactivation. Induction of immediate-early (ie) gene expression occurs in explants after approximately 9 days in culture and virus reactivation follows induction of ie gene expression with kinetics similar to that of productive infection in vitro. This occurs independently of TNF receptor signalling. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A results in more rapid induction of ie gene expression and reactivation of virus. Despite these results, which suggest a role for DNA methylation in maintenance of viral latency, we find that the major immediate-early promoter/enhancer is not methylated in latently infected mice. Our results support the hypothesis that latency is maintained by epigenetic control of ie gene expression, and that induction of ie gene expression leads to reactivation of virus, but suggest that these are not controlled by DNA methylation.

  11. Targeting the unique methylation pattern of androgen receptor (AR) promoter in prostate stem/progenitor cells with 5-aza-2'-deoxycytidine (5-AZA) leads to suppressed prostate tumorigenesis.

    PubMed

    Tian, Jing; Lee, Soo Ok; Liang, Liang; Luo, Jie; Huang, Chiung-Kuei; Li, Lei; Niu, Yuanjie; Chang, Chawnshang

    2012-11-16

    Androgen receptor (AR) expression surveys found that normal prostate/prostate cancer (PCa) stem/progenitor cells, but not embryonic or mesenchymal stem cells, expressed little AR with high methylation in the AR promoter. Mechanism dissection revealed that the differential methylation pattern in the AR promoter could be due to differential expression of methyltransferases and binding of methylation binding protein to the AR promoter region. The low expression of AR in normal prostate/PCa stem/progenitor cells was reversed after adding 5-aza-2'-deoxycytidine, a demethylating agent, which could then lead to decreased stemness and drive cells into a more differentiated status, suggesting that the methylation in the AR promoter of prostate stem/progenitor cells is critical not only in maintaining the stemness but also critical in protection of cells from differentiation. Furthermore, induced AR expression, via alteration of its methylation pattern, led to suppression of the self-renewal/proliferation of prostate stem/progenitor cells and PCa tumorigenesis in both in vitro assays and in vivo orthotopic xenografted mouse studies. Taken together, these data prove the unique methylation pattern of AR promoter in normal prostate/PCa stem/progenitor cells and the influence of AR on their renewal/proliferation and differentiation. Targeting PCa stem/progenitor cells with alteration of methylated AR promoter status might provide a new potential therapeutic approach to battle PCa because the PCa stem/progenitor cells have high tumorigenicity.

  12. Distinctive Roles of 5-aza-2′-deoxycytidine in Anterior Agranular Insular and Basolateral Amygdala in Reconsolidation of Aversive Memory Associated with Morphine in Rats

    PubMed Central

    Liu, Peng; Zhang, JianJun; Li, Ming; Sui, Nan

    2016-01-01

    5-aza-2′-deoxycytidine (5-aza), an inhibitor of DNA methyltransferases (DNMTs), has been implicated in aversive memory and the function of brain region involved in processing emotion. However, little is known about the role of 5-aza in the reconsolidation of opiate withdrawal memory. In the present study, using the morphine-naloxone induced conditioned place aversion (CPA) model in rats, we injected 5-aza into agranular insular (AI), granular insular (GI), basolateral amygdala (BLA) and central amygdala (CeA) immediately after the memory retrieval and tested the behavioral consequences at 24 h, 7 and 14 days after retrieval test. We found that 5-aza injection into AI disrupted the reconsolidation of morphine-associated withdrawal memory, but 5-aza injection into GI had no impact on the reconsolidation. Meanwhile, 5-aza injection into BLA but not CeA attenuated the withdrawal memory trace 14 days later. However, 5-aza administration to rats, in the absence of memory reactivation, had no effect on morphine-associated withdrawal memory. These findings suggest that 5-aza interferes with the reconsolidation of opiate withdrawal memory, and the roles of insular and amygdala in reconsolidation are distinctive. PMID:27014010

  13. Gelatinases-stimuli nanoparticles encapsulating 5-fluorouridine and 5-aza-2'-deoxycytidine enhance the sensitivity of gastric cancer cells to chemical therapeutics.

    PubMed

    Wu, Feng-lei; Li, Ru-Tian; Yang, Mi; Yue, Guo-Feng; Wang, Hui-yu; Liu, Qin; Cui, Fang-bo; Wu, Pu-yuan; Ding, Hui; Yu, Li-Xia; Qian, Xiao-Ping; Liu, Bao-Rui

    2015-07-10

    Aberrant methylation of the transcription factor AP-2 epsilon (TFAP2E) has been attributed to 5-fluorouridine (5-FU) sensitivity. 5-Aza-2'-deoxycytidine (DAC), an epigenetic drug that inhibits DNA methylation, is able to cause reactive expression of TFAP2E by demethylating activity. This property might be useful in enhancing the sensitivity of cancer cells to 5-FU. However, the effect of DAC is transient because of its instability. Here, we report the use of intelligent gelatinases-stimuli nanoparticles (NPs) to coencapsulate and deliver DAC and 5-FU to gastric cancer (GC) cells. The results showed that NPs encapsulating DAC, 5-FU, or both could be effectively internalized by GC cells. Furthermore, we found that the NPs enhanced the stability of DAC, resulting in improved re-expression of TFAP2E. Thus, the incorporation of DAC into NPs significantly enhanced the sensitivity of GC cells to 5-FU by inhibiting cell growth rate and inducing cell apoptosis. In conclusion, the results of this study clearly demonstrated that the gelatinases-stimuli NPs are an efficient means to simultaneously deliver epigenetic and chemotherapeutic drugs that may effectively inhibit cancer cell proliferation.

  14. Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia

    PubMed Central

    Garcia-Manero, Guillermo; Kantarjian, Hagop M.; Sanchez-Gonzalez, Blanca; Yang, Hui; Rosner, Gary; Verstovsek, Srdan; Rytting, Michael; Wierda, William G.; Ravandi, Farhad; Koller, Charles; Xiao, Lianchun; Faderl, Stefan; Estrov, Zeev; Cortes, Jorge; O'Brien, Susan; Estey, Elihu; Bueso-Ramos, Carlos; Fiorentino, Jackie; Jabbour, Elias; Issa, Jean-Pierre

    2006-01-01

    We conducted a phase 1/2 study of the combination of 5-aza-2′-deoxycytidine (decitabine) and the histone deacetylase inhibitor valproic acid (VPA) in patients with advanced leukemia, including older untreated patients. A group of 54 patients were treated with a fixed dose of decitabine (15 mg/m2 by IV daily for 10 days) administered concomitantly with escalating doses of VPA orally for 10 days. A 50 mg/kg daily dose of VPA was found to be safe. Twelve (22%) patients had objective response, including 10 (19%) complete remissions (CRs), and 2 (3%) CRs with incomplete platelet recovery (CRp). Among 10 elderly patients with acute myelogenous leukemia or myelodysplastic syndrome, 5 (50%) had a response (4CRs, 1CRp's). Induction mortality was observed in 1 (2%) patient. Major cytogenetic response was documented in 6 of 8 responders. Remission duration was 7.2 months (range, 1.3-12.6+ months). Overall survival was 15.3 months (range, 4.6-20.2+ months) in responders. Transient DNA hypomethylation and global histone H3 and H4 acetylation were induced, and were associated with p15 reactivation. Patients with lower pretreatment levels of p15 methylation had a significantly higher response rate. In summary, this combination of epigenetic therapy in leukemia was safe and active, and was associated with transient reversal of aberrant epigenetic marks. This trial was registered at www.clinicaltrials.gov as #NCT00075010. PMID:16882711

  15. Demethylation drug 5-Aza-2′-deoxycytidine-induced upregulation of miR-200c inhibits the migration, invasion and epithelial-mesenchymal transition of clear cell renal cell carcinoma in vitro

    PubMed Central

    JIANG, JUAN; YI, BO; DING, SIQING; SUN, JIAN; CAO, WEI; LIU, MENGZI

    2016-01-01

    The microRNA (miR)-200 family has been found to be involved in the process of mesenchymal-epithelial transition during renal development. Deregulation of miR-200c has been suggested to be involved in clear cell renal cell carcinoma (ccRCC). However, the precise role of miR-200c in the regulation of ccRCC metastasis has not been previously reported. In the present study, it was observed that miR-200c was frequently downregulated in ccRCC tissue compared with matched adjacent normal tissue. The expression of miR-200c was additionally reduced in ccRCC cell lines when compared with levels in normal renal cells. The DNA demethylation drug 5-Aza-2′-deoxycytidine (Aza) was used to treat several ccRCC cell lines, and it was observed that the expression of miR-200c was significantly increased following Aza treatment. Furthermore, treatment with Aza markedly inhibited ccRCC cell invasion and migration, while treatment with miR-200c inhibitor significantly enhanced invasion and migration of ccRCC cells. In addition, Aza treatment significantly promoted expression of E-cadherin and inhibited the expression of N-cadherin, while the inhibition of miR-200c downregulated E-cadherin and upregulated the expression of N-cadherin, suggesting that miR-200c has a suppressive role in epithelial-mesenchymal transition (EMT) of ccRCC cells. In conclusion, it was suggested that demethylation drug Aza-induced upregulation of miR-200c may inhibit migration, invasion and EMT in ccRCC cells. PMID:27123083

  16. Cytotoxicity of 5-Aza-2'-deoxycytidine against gastric cancer involves DNA damage in an ATM-P53 dependent signaling pathway and demethylation of P16(INK4A).

    PubMed

    Liu, Juan; Xie, Yi-Shan; Wang, Fu-Liang; Zhang, Li-Jun; Zhang, Yan; Luo, He-Sheng

    2013-02-01

    The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has increasingly attracted worldwide attention for its antineoplastic potential. The cytotoxitic mechanisms, however, especially, the relative contribution of silenced genes reactivation by demethylation and enzyme-DNA adduct formation to the efficacy of 5-Aza-CdR is still a crucial unresolved question. In this investigation, we demonstrated that 5-Aza-CdR treatment resulted in growth suppression in a concentration and time-dependent manner and G2 phrase arrest - hallmarks of a DNA damage response in gastric cancer AGS cells. Formation of DNA double-strand breaks, as monitored by comet assay was examined in an ATM (ataxia-telangiectasia mutated)-dependent manner based on the fact that PI3K inhibitor Wortmannin abolished the action of cytotoxicity of 5-Aza-CdR. Upon treatment with 5-Aza-CdR, ATM activation was clearly associated with P53 phosphorylation at Ser(15), which was directly responsible for 5-Aza-CdR modified P21(Waf1/Cip1) expression. Further exploration revealed that demethylation of P16(INK4A) correlated with the strikingly down-regulated expressions of DNA methyltransferase 3A as well as 3B was, at least in part, attributed to the cytotoxicity of 5-Aza-CdR in AGS cells. Conclusively, these results greatly enhance our understanding of the mechanisms of cytotoxicity of 5-Aza-CdR and strongly provide the preclinical rationale for an assessment of 5-Aza-CdR to ameliorate patient outcome with gastric cancer. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  17. 5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

    PubMed

    Wozniak, R J; Klimecki, W T; Lau, S S; Feinstein, Y; Futscher, B W

    2007-01-04

    The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2'-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

  18. All-trans retinoic acid enhances the effect of 5-aza-2'-deoxycytidine on p16INK4a demethylation, and the two drugs synergistically activate retinoic acid receptor β gene expression in the human erythroleukemia K562 cell line.

    PubMed

    Xiang, Lili; Dong, Weimin; Wang, Rong; Wei, Jiang; Qiu, Guoqiang; Cen, Jiannong; Chen, Zixing; Zheng, Xiao; Hu, Shaoyan; Xie, Xiaobao; Cao, Xiangshan; Gu, Weiying

    2014-07-01

    The aim of the current study was to investigate the antineoplastic activities of 5-aza-2'-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor β (RAR-β). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-β genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-β genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-β promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-β gene, which led to the upregulation of the RAR-β expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.

  19. Inhibition of DNA and Histone Methylation by 5-Aza-2′-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells

    PubMed Central

    Momparler, Richard L.; Côté, Sylvie; Momparler, Louise F.; Idaghdour, Youssef

    2017-01-01

    Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2′-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that

  20. Quantitation of inhibition of DNA methylation of the retinoic acid receptor beta gene by 5-Aza-2'-deoxycytidine in tumor cells using a single-nucleotide primer extension assay.

    PubMed

    Bovenzi, V; Momparler, R L

    2000-05-15

    The expression of several cancer-related genes has been reported to be silenced by DNA methylation of their promoter region. 5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactivate the in vitro expression of these genes. In future clinical trials in tumor therapy with 5-AZA-CdR a method to quantitate its inhibition of methylation of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue. We have modified the methylation-sensitive single-nucleotide primer extension assay reported by Gonzalgo and Jones (Nucleic Acids Res. 25, 2529-2531, 1997). Genomic DNA was treated with bisulfite and a fragment of the promoter region of the human retinoic acid receptor beta (RARbeta) gene, a tumor suppressor gene, was amplified using seminested PCR. Using two different primers we quantitated the inhibition of methylation produced by 5-AZA-CdR at two specific CpG sites in the RARbeta promoter in a human colon and a breast carcinoma cell line. The results obtained with the modified assay show a precise and reproducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells.

  1. Elevated fetal haemoglobin is a predictor of better outcome in MDS/AML patients receiving 5-aza-2'-deoxycytidine (Decitabine).

    PubMed

    Lübbert, Michael; Ihorst, Gabriele; Sander, Philipp N; Bogatyreva, Ljudmila; Becker, Heiko; Wijermans, Pierre W; Suciu, Stefan; Bissé, Emmanuel; Claus, Rainer

    2017-02-01

    Although azanucleoside DNA-hypomethylating agents (HMAs) are routinely used for the treatment of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), very few outcome predictors have been established. Expression of the β-like globin gene locus is tightly regulated by DNA methylation, is HMA-sensitive in vitro, and fetal haemoglobin (HbF) expression is under study as a potential biomarker for response of MDS patients to azacitidine. We determined HbF expression in 16 MDS and 36 AML patients receiving decitabine (DAC). Pre-treatment HbF was already elevated (>1·0% of total haemoglobin) in 7/16 and 12/36 patients, and HbF was induced by DAC in 81%/54% of MDS/AML patients, respectively. Elevated pre-treatment HbF was associated with longer median overall survival (OS): 26·6 vs. 8·6 months for MDS (hazard ratio [HR] 8·56, 95% confidence interval [CI] 1·74-42·49, P = 0·008, with similarly longer progression-free and AML-free survival), and 10·0 vs. 2·9 months OS for AML (HR 3·01, 95% CI 1·26-7·22, P = 0·014). In a multivariate analysis, the prognostic value of HbF was retained. Time-dependent Cox models revealed that the prognostic value of treatment-induced HbF induction was inferior to that of pre-treatment HbF. In conclusion, we provide first evidence for in vivo HbF induction by DAC in MDS/AML, and demonstrate prognostic value of elevated pre-treatment HbF, warranting prospective, randomized studies.

  2. Inhibition of ribonucleotide reductase by 2'-substituted deoxycytidine analogs: possible application in AIDS treatment.

    PubMed Central

    Bianchi, V; Borella, S; Calderazzo, F; Ferraro, P; Chieco Bianchi, L; Reichard, P

    1994-01-01

    After phosphorylation to the corresponding diphosphates, 2'-azido-2'-deoxycytidine and 2'-difluorocytidine act as powerful inhibitors of ribonucleotide reductase. Phosphorylation requires deoxycytidine kinase, an enzyme with particularly high activity in lymphoid cells. Therefore, the deoxycytidine analogs can be expected to inhibit the reductase with some specificity for the lymphoid system. Pretreatment of human CEM lymphoblasts with the analogs considerably increased the phosphorylation of 3'-deoxy-3'-azidothymidine (AzT). The increased phosphorylation of AzT is caused by a prolongation of the S phase of the cell cycle. Our results suggest the possibility of a combination of 2'-substituted deoxycytidine analogs with AzT in the treatment of AIDS. Gao et al. [Gao, W.-Y., Cara, A., Gallo, R. C. & Lori, F. (1993) Proc. Natl. Acad. Sci. USA 90, 8925-8928] have suggested the use of the ribonucleotide reductase inhibitor hydroxyurea for this purpose, since the resulting decrease in the size of deoxyribonucleotide pools decreases the processivity of the HIV reverse transcriptase. From our results it would appear that the 2'-substituted deoxycytidine analogs might be preferable to hydroxyurea. PMID:8078894

  3. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies black-footed cat cloned embryos.

    PubMed

    Gómez, M C; Biancardi, M N; Jenkins, J A; Dumas, C; Galiguis, J; Wang, G; Earle Pope, C

    2012-12-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  4. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

    USGS Publications Warehouse

    Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

    2012-01-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  5. 2'-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2'-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites.

    PubMed

    Lamparska, Katarzyna; Clark, Jarrod; Babilonia, Gail; Bedell, Victoria; Yip, Wesley; Smith, Steven S

    2012-10-01

    5-Aza-2'-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2'-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.

  6. 2′-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2′-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites

    PubMed Central

    Lamparska, Katarzyna; Clark, Jarrod; Babilonia, Gail; Bedell, Victoria; Yip, Wesley; Smith, Steven S.

    2012-01-01

    5-Aza-2′-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2′-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product. PMID:22850746

  7. Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor, target, transition state structure and enzyme conformation.

    PubMed

    Brank, Adam S; Eritja, Ramon; Garcia, Ramon Guimil; Marquez, Victor E; Christman, Judith K

    2002-10-11

    The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.

  8. All-trans retinoic acid combined with 5-Aza-2 Prime -deoxycitidine induces C/EBP{alpha} expression and growth inhibition in MLL-AF9-positive leukemic cells

    SciTech Connect

    Fujiki, Atsushi; Imamura, Toshihiko; Sakamoto, Kenichi; Kawashima, Sachiko; Yoshida, Hideki; Hirashima, Yoshifumi; Miyachi, Mitsuru; Yagyu, Shigeki; Nakatani, Takuya; Sugita, Kanji; Hosoi, Hajime

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer We tested whether ATRA and 5-Aza affect AML cell differentiation and growth. Black-Right-Pointing-Pointer Cell differentiation and growth arrest were induced in MLL-AF9-expressing cells. Black-Right-Pointing-Pointer Increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1 were also observed. Black-Right-Pointing-Pointer MLL-AF4/AF5q31-expressing cells are less sensitive to ATRA and 5-Aza. Black-Right-Pointing-Pointer Different MLL fusion has distinct epigenetic properties related to RA pathway. -- Abstract: The present study tested whether all-trans retinoic acid (ATRA) and 5-Aza-2 Prime -deoxycitidine (5-Aza) affect AML cell differentiation and growth in vitro by acting on the CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) and c-Myc axis. After exposure to a combination of these agents, cell differentiation and growth arrest were significantly higher in human and murine MLL-AF9-expressing cells than in MLL-AF4/AF5q31-expressing cells, which were partly associated with increased expression of C/EBP{alpha}, C/EBP{epsilon}, and PU.1, and decreased expression of c-Myc. These findings indicate that MLL-AF9-expressing cells are more sensitive to ATRA and 5-Aza, indicating that different MLL fusion proteins possess different epigenetic properties associated with retinoic acid pathway inactivation.

  9. Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.

    PubMed

    Yerxa, B R; Sabater, J R; Davis, C W; Stutts, M J; Lang-Furr, M; Picher, M; Jones, A C; Cowlen, M; Dougherty, R; Boyer, J; Abraham, W M; Boucher, R C

    2002-09-01

    INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt] is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y(2) receptor. In primate lung tissues, the P2Y(2) receptor mRNA was located by in situ hybridization predominantly in epithelial cells and not in smooth muscle or stromal tissue. The pharmacologic profile of INS37217 parallels that of UTP, leading to increased chloride and water secretion, increased cilia beat frequency, and increased mucin release. The combined effect of these actions was confirmed in an animal model of tracheal mucus velocity that showed that a single administration of INS37217 significantly enhanced mucus transport for at least 8 h after dosing. This extended duration of action is consistent with the ability of INS37217 to resist metabolism by airway cells and sputum enzymes. The enhanced metabolic stability and resultant increased duration of improved mucociliary clearance may confer significant advantages to INS37217 over other P2Y(2) agonists in the treatment of diseases such as cystic fibrosis.

  10. Continuous Zebularine Treatment Effectively Sustains Demethylation in Human Bladder Cancer Cells

    PubMed Central

    Cheng, Jonathan C.; Weisenberger, Daniel J.; Gonzales, Felicidad A.; Liang, Gangning; Xu, Guo-Liang; Hu, Ye-Guang; Marquez, Victor E.; Jones, Peter A.

    2004-01-01

    During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5′ region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2′-deoxycytidine followed by zebularine hindered the remethylation of the p16 5′ region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen. PMID:14729971

  11. Induction of a specific CD8+ T-cell response to cancer/testis antigens by demethylating pre-treatment against osteosarcoma.

    PubMed

    Li, Binghao; Zhu, Xiaobing; Sun, Lingling; Yuan, Li; Zhang, Jian; Li, Hengyuan; Ye, Zhaoming

    2014-11-15

    Conventional non-surgical therapeutic regimens against osteosarcoma are subject to chemoresistance and tumor relapse, and immunotherapy may be promising for this tumor. However, it's hard to find satisfactory epitopes for immunotherapy against osteosarcoma. Cancer/testis antigens (CTAs), such as MAGE-A family and NY-ESO-1, the potential antigens that almost exclusively express in tumor cells and immune-privileged sites, have been found expressed in osteosarcoma also. Nevertheless, the expression of CTAs is downregulated in many tumors, constraining the application of immunotherapy. In this article, we demonstrate that the expression of MAGE-A family and NY-ESO-1 in osteosarcoma cells can be upregulated following treatment with demethylating agent 5-aza-2'-deoxycytidine and consequently induces a CTA specific CD8+ T-cell response against osteosarcoma in vitro and in vivo. The in vivo imaging was realized by using luciferase-transfected HOS cells and DiR labeled T-cells in severely combined immunodeficiency mouse models. Cytotoxic T cells specifically recognizing MAGE-A family and NY-ESO-1 clustered at the tumor site in mice pre-treated with DAC and resulted in tumor growth suppression, while it was not observed in mice without DAC pre-treatment. This study is important for more targeted therapeutic approaches and suggests that adoptive immunotherapy, combined with demethylating treatment, has the potential for non-surgical therapeutic strategy against osteosarcoma.

  12. Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer.

    PubMed

    Scholpa, N E; Kolli, R T; Moore, M; Arnold, R D; Glenn, T C; Cummings, B S

    2016-10-25

    This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

  13. Chemical synthesis of 2'-deoxyoligonucleotides containing 5-fluoro-2'-deoxycytidine.

    PubMed Central

    Schmidt, S; Pein, C D; Fritz, H J; Cech, D

    1992-01-01

    2'-Deoxyoligonucleotides with 5-fluorocytosine residues incorporated at specific positions of the nucleotide sequence are tools of great potential in the study of the catalytic mechanism by which DNA cytosine methyltransferases methylate the 5-position of DNA cytosine residues in specific sequence contexts. Chemical synthesis of such oligonucleotides is described. Two alternative approaches have been developed, one of which proceeds via a fully protected phosphoramidite of 5-fluoro-4-methylmercapto-2'-deoxyuridine 2, the other via a fully protected phosphoramidite of 5-fluoro-2'-deoxycytidine 3. Either building block can be used in automated oligonucleotide synthesis applying standard elongation cycles and deprotection procedures exclusively. The methylmercapto function of 2 is replaced by an amino group in the final ammonia treatment used for cleavage from support and base deprotection. PMID:1598200

  14. Assignment of the human deoxycytidine kinase (DCK) gene to chromosome 4 band q13. 3-q21. 1

    SciTech Connect

    Stegmann, A.P.A.; Honders, M.W.; Bolk, M.W.J.; Willemze, R.; Landegent, J.E.; Wessels, J. )

    1993-08-01

    The enzyme deoxycytidine kinase (DCK) is the key enzyme of the salvage pathway for pyrimidine synthesis. It is responsible for the phosphorylation of deoxycytidine and several deoxycytidine analogues that are used as antimetabolites in the treatment of human cancers. For instance, the cytotoxic activity of 1-[beta]-D-arabinofuranosylcytosine (AraC), used in the chemotherapy of acute myeloid leukemia (AML), is dependent on its phosphorylation by DCK. The occurrence of clinical AraC resistance, which is usually marked by functional DCK deficiency, is one of the major obstacles in the successful treatment of AML. The cDNA sequence of the DCK gene was published and, more recently, mutational inactivation of the DCK gene has been described as a possible cause of DCK deficiency. In this study the authors report on the chromosomal localization of the DCK gene by means of fluorescence in situ hybridization. 6 refs., 2 figs.

  15. Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

    PubMed

    Gay, Maresha S; Li, Yong; Xiong, Fuxia; Lin, Thant; Zhang, Lubo

    2015-01-01

    The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

  16. Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells

    PubMed Central

    2014-01-01

    Background Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. Methods We analyzed the effect of treatment with the cytosine analogue 5 aza-2deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. Conclusions Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications. PMID:24795534

  17. Radioimmunoassays of plasma thymidine, uridine, deoxyuridine, and cytidine/deoxycytidine

    SciTech Connect

    Dudman, N.P.B.; Deveski, W.B.; Tattersall, M.H.N.

    1981-08-01

    Radioimmunoassay techniques have been developed for the assay of thymidine, uridine, deoxyuridine, and deoxycytidine. Plasma levels of the first three nucleosides have been measured, and an upper limit has been determined for the plasma concentration of deoxycytidine. The assays involve displacement of a (3H)pyrimidine nucleoside from the appropriate labeled rabbit immunoglobulin. By assaying a mixture of uridine and deoxyuridine in the presence and absence of borax, the concentrations of both nucleosides have been measured. In seven healthy adults, plasma levels of uridine were 21.1 +/- 8.4 ..mu..M (mean +/- SD) and of deoxyuridine were 0.62 +/- 0.39 ..mu..M. In cancer patients, thymidine levels were 7.5 +/- 2.7 x 10/sup -7/M. The upper limit for plasma deoxycytidine levels in six healthy adults was 0.71 +/- 0.1 ..mu..M.

  18. Identification of 4-(3-Pyridyl)-4-oxobutyl-2′-deoxycytidine Adducts Formed in the Reaction of DNA with 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A Chemically Activated Form of Tobacco-Specific Carcinogens

    PubMed Central

    2017-01-01

    Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N′-nitrosonornicotine (NNN, 2) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2′-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2′-deoxycytidine and 2′-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2′-deoxycytidine adduct, identified as its stable cytosine analogue O2-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (12), was O2-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (13), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (14) and N4-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (15) were also observed. The potential conversion of relatively unstable 2′-deoxycytidine adducts to stable 2′-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2, and demonstrate the presence of previously unidentified 2′-deoxycytidine adducts in this DNA. PMID:28393135

  19. Identification of 4-(3-Pyridyl)-4-oxobutyl-2'-deoxycytidine Adducts Formed in the Reaction of DNA with 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A Chemically Activated Form of Tobacco-Specific Carcinogens.

    PubMed

    Michel, Anna K; Zarth, Adam T; Upadhyaya, Pramod; Hecht, Stephen S

    2017-03-31

    Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N'-nitrosonornicotine (NNN, 2) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2'-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2'-deoxycytidine and 2'-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2'-deoxycytidine adduct, identified as its stable cytosine analogue O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (12), was O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (13), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (14) and N(4)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine (15) were also observed. The potential conversion of relatively unstable 2'-deoxycytidine adducts to stable 2'-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2, and demonstrate the presence of previously unidentified 2'-deoxycytidine adducts in this DNA.

  20. Role of histone modifications and DNA methylation in the regulation of O6-methylguanine-DNA methyltransferase gene expression in human stomach cancer cells.

    PubMed

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2010-05-01

    To determine a possible function of histone modifications in stomach carcinogenesis, we analyzed global and MGMT-promoter levels of di-methyl-H3-K9, di-methyl-H3-K4 and acetyl-H3-K9, as well as MGMT DNA methylation and mRNA expression following treatment with 5-aza-2' -deoxycytidine and/or Trichostatin A. We found that histone H3-K9 di-methylation, H3-K4 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence MGMT. The results indicate that histone modifications as well as DNA methylation may be involved in stomach carcinogenesis. In addition to its effect on DNA methylation, 5-aza-2' -deoxycytidine can act at histone modification level to reactivate MGMT expression in a region-specific and DNA methylation-dependent manner.

  1. Preclinical studies of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in pediatric brain tumors.

    PubMed

    Morfouace, Marie; Nimmervoll, Birgit; Boulos, Nidal; Patel, Yogesh T; Shelat, Anang; Freeman, Burgess B; Robinson, Giles W; Wright, Karen; Gajjar, Amar; Stewart, Clinton F; Gilbertson, Richard J; Roussel, Martine F

    2016-01-01

    Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.

  2. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

    PubMed Central

    Kim, Woosuk; Le, Thuc M.; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T.; Abt, Evan R.; Capri, Joseph R.; Austin, Wayne R.; Van Valkenburgh, Juno S.; Steele, Dalton; Gipson, Raymond M.; Slavik, Roger; Cabebe, Anthony E.; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S.; Lee, Jason T.; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F.; Witte, Owen N.; Donahue, Timothy R.; Phelps, Michael E.; Herschman, Harvey R.; Herrmann, Ken; Czernin, Johannes; Radu, Caius G.

    2016-01-01

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds—[18F]Clofarabine; 2-chloro-2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-adenine ([18F]CFA) and 2′-deoxy-2′-[18F]fluoro-9-β-d-arabinofuranosyl-guanine ([18F]F-AraG)—for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [18F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [18F]F-AraG is a better substrate for dGK than for dCK. [18F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [18F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [18F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [18F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [18F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [18F]CFA PET as a new cancer biomarker for treatment stratification and monitoring. PMID:27035974

  3. [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity.

    PubMed

    Kim, Woosuk; Le, Thuc M; Wei, Liu; Poddar, Soumya; Bazzy, Jimmy; Wang, Xuemeng; Uong, Nhu T; Abt, Evan R; Capri, Joseph R; Austin, Wayne R; Van Valkenburgh, Juno S; Steele, Dalton; Gipson, Raymond M; Slavik, Roger; Cabebe, Anthony E; Taechariyakul, Thotsophon; Yaghoubi, Shahriar S; Lee, Jason T; Sadeghi, Saman; Lavie, Arnon; Faull, Kym F; Witte, Owen N; Donahue, Timothy R; Phelps, Michael E; Herschman, Harvey R; Herrmann, Ken; Czernin, Johannes; Radu, Caius G

    2016-04-12

    Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  4. Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs

    PubMed Central

    Elmehriki, Adam AH; Suchý, Mojmír; Chicas, Kirby J; Wojciechowski, Filip; Hudson, Robert HE

    2014-01-01

    Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2’-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2’-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2’-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior. PMID:25483932

  5. Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs.

    PubMed

    Elmehriki, Adam A H; Suchý, Mojmír; Chicas, Kirby J; Wojciechowski, Filip; Hudson, Robert H E

    2014-01-01

    Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2'-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2'-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2'-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior.

  6. DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins.

    PubMed

    Klasić, Marija; Krištić, Jasminka; Korać, Petra; Horvat, Tomislav; Markulin, Dora; Vojta, Aleksandar; Reiding, Karli R; Wuhrer, Manfred; Lauc, Gordan; Zoldoš, Vlatka

    2016-04-13

    Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

  7. DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins

    PubMed Central

    Klasić, Marija; Krištić, Jasminka; Korać, Petra; Horvat, Tomislav; Markulin, Dora; Vojta, Aleksandar; Reiding, Karli R.; Wuhrer, Manfred; Lauc, Gordan; Zoldoš, Vlatka

    2016-01-01

    Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2′-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene. PMID:27073020

  8. Synthesis and properties of 2'-deoxycytidine triphosphate carrying c-myc tag sequence.

    PubMed

    Hinz, M; Gottschling, D; Eritja, R; Seliger, H

    2000-01-01

    The synthesis of 2'-deoxycytidine triphosphate carrying mercaptoethyl groups at position 4 of cytosine is described. This nucleoside triphosphate was reacted with a maleimido-peptide carrying the c-myc tag-sequence to yield a peptide-nucleoside triphosphate chimera. Primer extension studies showed that the nucleoside triphosphate modified with the peptide sequence is incorporated by DNA polymerases opposite guanine.

  9. Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

    PubMed

    Shibu, Marthandam Asokan; Yang, Hsueh-Hui; Lo, Chaur-Tsuen; Lin, Hong-Shin; Liu, Shu-Ying; Peng, Kou-Cheng

    2014-02-26

    Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.

  10. The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death

    PubMed Central

    Zhong, Rui; Xin, Rui; Chen, Zongyan; Liang, Nan; Liu, Yang; Ma, Shumei; Liu, Xiaodong

    2016-01-01

    Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway. PMID:27879648

  11. Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease

    PubMed Central

    Weng, Tingting; Karmouty-Quintana, Harry; Garcia-Morales, Luis J.; Molina, Jose G.; Pedroza, Mesias; Bunge, Raquel R.; Bruckner, Brian A.; Loebe, Matthias; Seethamraju, Harish; Blackburn, Michael R.

    2013-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by persistent inflammation and tissue remodeling and is a leading cause of death in the United States. Increased apoptosis of pulmonary epithelial cells is thought to play a role in COPD development and progression. Identification of signaling pathways resulting in increased apoptosis in COPD can be used in the development of novel therapeutic interventions. Deoxyadenosine (dAdo) is a DNA breakdown product that amplifies lymphocyte apoptosis by being phosphorylated to deoxyadenosine triphosphate (dATP). dAdo is maintained at low levels by adenosine deaminase (ADA). This study demonstrated that mice lacking ADA developed COPD manifestations in association with elevated dAdo and dATP levels and increased apoptosis in the lung. Deoxycitidine kinase (DCK), a major enzyme for dAdo phosphorylation, was up-regulated in mouse and human airway epithelial cells in association with air-space enlargement. Hypoxia was identified as a novel regulator of DCK, and inhibition of DCK resulted in diminished dAdo-mediated apoptosis in the lungs. Our results suggest that activating the dAdo-DCK-dATP pathway directly results in increased apoptosis in the lungs of mice with air-space enlargement and suggests a novel therapeutic target for the treatment of COPD.—Weng, T., Karmouty-Quintana, H., Garcia-Morales, L. J., Molina, J. G., Pedroza, M., Bunge, R. R., Bruckner, B. A., Loebe, M., Seethamraju, H., and Blackburn, M. R. Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease. PMID:23392349

  12. The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death.

    PubMed

    Zhong, Rui; Xin, Rui; Chen, Zongyan; Liang, Nan; Liu, Yang; Ma, Shumei; Liu, Xiaodong

    2016-11-21

    Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway.

  13. Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase

    PubMed Central

    Winter, Flore; Kretzschmar, Franziska K.; Brayé, Mélanie; Martin, Darren P.; Lener, Daniela; Negroni, Matteo

    2015-01-01

    Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches. PMID:26485161

  14. Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase.

    PubMed

    Coulibaly, Safiatou T; Rossolillo, Paola; Winter, Flore; Kretzschmar, Franziska K; Brayé, Mélanie; Martin, Darren P; Lener, Daniela; Negroni, Matteo

    2015-01-01

    Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.

  15. Genomic structure and chromosomal localization of the human deoxycytidine kinase gene

    SciTech Connect

    Song, J.J.; Walker, S.; Gribbin, T. ); Chen, E. Univ. of North Carolina, Chapel Hill ); Johnson, E.E.; Spychala, J.; Mitchell, B.S. )

    1993-01-15

    Deoxycytidine kinase (NTP:deoxycytidine 5[prime]-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5[prime] flanking regions and delinated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5[prime] flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5[prime] upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have >6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5[prime] sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression. 32 refs., 4 figs., 2 tabs.

  16. Nonenantioselectivity Property of Human Deoxycytidine Kinase Explained by Structures of the Enzyme in Complex with [subscript L]- and [subscript D]-Nucleosides

    SciTech Connect

    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Lavie, Arnon

    2008-07-31

    Biological molecules are predominantly enantioselective. Yet currently two nucleoside analogue prodrugs (3TC and FTC) with opposite chirality compared to physiological nucleosides are clinically approved for the treatment of HIV infections. These prodrugs require conversion to their triphosphorylated forms to achieve pharmacological activity. The first step in the activation of these agents is catalyzed by human deoxycytidine kinase (dCK). This enzyme possesses the ability to phosphorylate nucleosides of the unnatural L-chirality. To understand the molecular basis for the nonenantioselectivity of dCK, we solved the crystal structures of the enzyme in complex with the L-enantiomer and of its physiological substrate deoxycytidine and with the L-nucleoside analogue FTC. These were compared to a structure solved with D-dC. Our results highlight structural adjustments imposed on the L-nucleosides and properties of the enzyme endowing it with the ability to phosphorylate substrates with nonphysiological chirality. This work reveals the molecular basis for the activation of L-nucleosides by dCK.

  17. The non-enantioselectivity property of human deoxycytidine kinase explained by structures of the enzyme in complex with l- and d-nucleosides

    PubMed Central

    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Lavie, Arnon

    2008-01-01

    Biological molecules are predominantly enantioselective. Yet, currently two nucleoside analog prodrugs (3TC and FTC) with opposite chirality to physiological nucleosides are clinically approved for the treatment of HIV infections. These prodrugs require conversion to their tri-phosphorylated forms to achieve pharmacological activity. The first step in the activation of these agents is catalyzed by human deoxycytidine kinase (dCK). This enzyme possesses the ability to phosphorylate nucleosides of the unnatural l-chirality. To understand the molecular basis for the non-enantioselectivity of dCK we solved the crystal structures of the enzyme in complex with the l-enantiomer of its physiological substrate deoxycytidine and with the l-nucleoside analog FTC. These were compared to a structure solved with d-dC. Our results highlight structural adjustments imposed on the l-nucleosides, and properties of the enzyme endowing it with the ability to phosphorylate substrates with non-physiological chirality. This work reveals the molecular basis for the activation of l-nucleosides by dCK. PMID:17530837

  18. Methylation of Integrin α4 and E-Cadherin Genes in Human Prostate Cancer.

    PubMed

    Mostafavi-Pour, Z; Kianpour, S; Dehghani, M; Mokarram, P; Torabinejad, S; Monabati, A

    2015-09-01

    Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

  19. [Decitabine treatment in myelodysplastic syndromes--results of a compassionate patient program in Israel].

    PubMed

    Klepfish, Abraham; Silbershatz, Itay; Lugassy, Gilles; Shimoni, Avichai; Mittelman, Moshe

    2013-10-01

    Hypermethylation of tumor suppressor genes (TSG) has been recognized as an important factor in the pathogenesis of malignancies, including myelodysplastic syndromes (MDS). Decitabine (trade name Dacogen; 5-aza-2'-deoxycytidine) is a cytosine analog which inhibits the enzyme DNA methyltransferase (DNMT), inducing hypomethylation and activates TSG, leading to tumor cell growth inhibition. In clinical trials with hypomethylating agents in advanced MDS, a total response rate of 30-73% has been observed, with a complete response (CR) of 9-37%, partial response (PR] of similar rate and a hematologic improvement (HI] in 20-48% of the patients. We report the results of a national Israeli compassionate program of decitabine administration to patients with advanced MDS. From July 2007 through August 2008, under the joint sponsorship of The Israel Society of Hematology and Blood Transfusions and Janssen, Israel, a compassionate program was conducted. Decitabine was administered to patients with advanced MDS who were not candidates for any other anti-MDS treatment, except for supportive care. The selected regimen was a 5-day intravenous administration of 20 mg/m/d, every 28 days. After the program had been completed, an approval of the institutional Helsinki committees was obtained, and the data were collected in an attempt to evaluate the results of this novel treatment. The standard response criteria, i.e. total response, CR, PR and HI were applied. Toxicity, survival and leukemic transformation rate were also analyzed. Twenty-four patients with advanced MDS participated in the program but evaluable information could be collected only on 17 patients. The median number of therapeutic cycles was two per patient. Twelve patients were transfusion-dependent at program onset, of whom 7 either benefited from reduced transfusion requirements or became transfusion-free. The overall response rate was 26%, with 23% PR and 13% HI. Two patients (13%) demonstrated leukemic transformation

  20. Atorvastatin treatment modulates p16 promoter methylation to regulate p16 expression.

    PubMed

    Zhu, Boqian; Gong, Yaoyao; Yan, Gaoliang; Wang, Dong; Wang, Qingjie; Qiao, Yong; Hou, Jiantong; Liu, Bo; Tang, Chengchun

    2017-06-01

    Intimal hyperplasia, the key event of arterial restenosis, is a result of cell proliferation and cell migration. Atorvastatin exerts an inhibitory effect on cell proliferation and migration, but the mechanism remains largely unknown. p16, as a well-known tumor suppressor, was also reported to suppress cell growth and migration, but with an unclear mechanism. In this study, we demonstrated that atorvastatin represses cell proliferation and migration in vascular smooth muscle cells (VSMCs) and that this process is mediated by p16. Furthermore, we found that DNA methylation in the p16 promoter was reduced and p16 expression was restored in VSMCs treated with 5-aza-2'-deoxycytidine or atorvastatin. However, the effect was absent when DNA methyltransferase 1 (DNMT1) was knocked down with RNA interference. These observations demonstrated that atorvastatin regulates p16 expression via DNMT1-induced DNA methylation in the p16 promoter. In addition, we found that the mitogen-activated protein kinase (MAPK) pathway was involved in the regulation of p16 by DNMT1, and MAPK inhibitors partially released the effects of atorvastatin on p16 and DNMT1. Finally, we illustrated that atorvastatin inhibits neointima formation and modulates p16 expression in balloon catheter-injured rat carotid artery. Taken together, we demonstrated that atorvastatin inhibits neointima formation through inducing p16 expression by affecting DNA methylation in the p16 promoter region. © 2017 Federation of European Biochemical Societies.

  1. Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

    SciTech Connect

    Kusakabe, Masashi; Watanabe, Kousuke; Emoto, Noriko; Aki, Naomi; Kage, Hidenori; Nagase, Takahide; Nakajima, Jun; Yatomi, Yutaka; Ohishi, Nobuya; Takai, Daiya

    2009-12-25

    We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2'-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2'-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2'-deoxycytidine.

  2. The interplay between epigenetic silencing, oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells.

    PubMed

    Swiderek, Ewelina; Kalas, Wojciech; Wysokinska, Edyta; Pawlak, Alicja; Rak, Janusz; Strzadala, Leon

    2013-11-29

    Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood. In the present study we describe profound effect of KRas on the expression of methylated BNIP3 in colorectal cancer cells and explore the interplay between HIF-1, hypoxia pathway and oncogenic KRas in this context. We observed that BNIP3 mRNA remains undetectable in aggressive DLD-1 cells harboring G13D mutant KRAS and HT-29 colorectal cancer cells unless the cells are exposed to demethylating agents such as 5-aza-2'-deoxycytidine. Following this treatment BNIP3 expression remains uniquely dependent on the Ras activity. We found that hypoxia or pharmacological activation of HIF-1 alone contributes to, but is not sufficient for efficient induction of BNIP3 mRNA transcription in cells lacking mutant KRas activity. The up-regulation of BNIP3 by KRas in this setting is mediated by the MAPK pathway, and is attenuated by the respective inhibitors (PD98059, U0126). Thus, we demonstrate the novel mechanism where activity of Ras is essential for 5-aza-2'-deoxycytidine-mediated BNIP3 expression. Moreover, we found that 5-aza-2'-deoxycytidine-mediated or enforced up-regulation of BNIP3 in DLD-1 cells results in KRas-dependent resistance to 5-Fluorouracil. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Fingerprinting DNA oxidation processes: IR characterization of the 5-methyl-2'-deoxycytidine radical cation.

    PubMed

    Bucher, Dominik B; Pilles, Bert M; Pfaffeneder, Toni; Carell, Thomas; Zinth, Wolfgang

    2014-02-24

    Methylated cytidine plays an important role as an epigenetic signal in gene regulation. Its oxidation products are assumed to be involved in active demethylation processes but also in damaging DNA. Here, we report the photochemical production of the 5-methyl-2'-deoxycytidine radical cation via a two-photon ionization process. The radical cation is detected by time-resolved IR spectroscopy and identified by band assignment using density functional theory calculations. Two final oxidation products are characterized with liquid chromatography coupled to mass spectrometry. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Indoxyl Sulfate Enhance the Hypermethylation of Klotho and Promote the Process of Vascular Calcification in Chronic Kidney Disease

    PubMed Central

    Chen, Jing; Zhang, Xiaoyan; Zhang, Han; Liu, Tongqiang; Zhang, Hui; Teng, Jie; Ji, Jun; Ding, Xiaoqiang

    2016-01-01

    Chronic kidney disease (CKD) is a state of Klotho deficiency. The Klotho expression may be suppressed due to DNA hypermethylation in cancer cells so we have investigated the effects and possible mechanisms by which Klotho expression is regulated in human aortic smooth muscle cells (HASMCs). The vascular Klotho hypermethylation in radial arteries of patients with end-stage renal disease was described. Cultured HASMCs and 5/6-nephrectomized Sprague Dawley (SD) rats treated with indoxyl sulfate (IS) were used as in vitro and in vivo models, respectively. IS increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in HASMCs, and potentiated HASMCs calcification. The expression of DNA methyltransferase (DNMT) 1 and 3a in HASMCs treated with IS was significantly increased and specific inhibition of DNA methyltransferase 1 by 5-aza-2'-deoxycytidine(5Aza-2dc) caused demethylation of the Klotho gene and increased Klotho expression. In rats, injection of IS potentiated vascular calcification, increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in the aortic medial layer and all of these changes could be reverted by 5Aza-2dc treatment. Transcriptional suppression of vascular Klotho gene expression by IS and epigenetic modification of Klotho by IS may be an important pathological mechanism of vascular calcification in CKD. PMID:27766038

  5. Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

    PubMed Central

    Bunimovich, Yuri L.; Nair-Gill, Evan; Riedinger, Mireille; McCracken, Melissa N.; Cheng, Donghui; McLaughlin, Jami; Radu, Caius G.; Witte, Owen N.

    2014-01-01

    Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy. PMID:25101980

  6. Base-pairing selectivity of a ureido-linked phenyl-2'-deoxycytidine derivative.

    PubMed

    Nakano, Shu-ichi; Oka, Hirohito; Yamaguchi, Daisuke; Fujii, Masayuki; Sugimoto, Naoki

    2012-12-28

    Incorporation of modified nucleotides into nucleic acid strands often produces conformational constraints and steric hindrances that may change the property of base pairing. In this study, we investigated a 2'-deoxycytidine derivative that tethers a phenyl moiety to the exocyclic amino group of cytosine linked through a ureido group. This derivative compound is structurally similar to the carbamoylated DNA base lesions produced in cells. The thermodynamic and structural studies showed that the modified dC formed the base pair with dG in the complementary strand, but the base-pairing selectivity toward dG was decreased under poly(ethylene glycol)-mediated osmotic stress. The phenyl group and the ureido linker attached to dC provided selectivity for the formation of base pairing exclusively with dG in a wide range of pH conditions, whereas unmodified dC stabilized the pairings with dA or dC in acidic solutions. Moreover, this modified base could not form self-pairing through intermolecular hydrogen bonds. We suggest that formation of weak pairing and protonation of the cytosine base are hindered due to the base modification. These data provide insights into the pairing selectivity of carbamoylated cytosine lesions produced in cells, and suggest applications of the 2'-deoxycytidine derivatives in medical technologies, molecular biology experiments, and synthesis of a supramolecular network of DNA strands.

  7. 5-Ethynyl-2'-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells.

    PubMed

    Qu, Dezhong; Wang, Guoxing; Wang, Zhe; Zhou, Li; Chi, Weilin; Cong, Shujie; Ren, Xiaoshuai; Liang, Peizhou; Zhang, Biliang

    2011-10-01

    The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [(3)H]thymidine or 5-bromo-2'-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via "click" chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2'-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.

  8. DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells.

    PubMed

    Liu, Jian; Zhu, Xinjiang; Xu, Xiaoyang; Dai, Dongqiu

    2014-01-01

    Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate tumor metastasis suppressor gene. Our study was to determine whether DNA hypermethylation and histone modification at the NGX6 gene promoter play important roles in silencing NGX6 expression in gastric cancer. NGX6 expression was downregulated in all gastric cancer cells and 76.19 % tissues. In three GC cell lines, hypermethylated NGX6 loci were characterized by histone H3-K9 hypoacetylation and hypermethylation. Trichostatin A treatment could moderately increase H3-K9 acetylation at the silenced loci; however, it had no effect on DNA and H3-K9 methylation and minimal effects on NGX6 expression. In contrast, 5'aza-2'-deoxycytidine treatment could rapidly decrease DNA and H3-K9 methylation at the silenced loci, leading to the reexpression of NGX6. Combined treatment with 5'aza-2'-deoxycytidine and trichostatin A had synergistic effects on the reexpression of NGX6 at the hypermethylation loci. Our current study shows that NGX6 expression is downregulated in GC cancer cells and tissues due to NGX6 promoter methylation and H3-K9 methylation, but not H3-K9 acetylation. Our findings indicate that the downregulation of NGX6 expression contributes to the development and progression of gastric cancer. More studies are needed to determine the precise mechanism of NGX6 in the progression of gastric cancer.

  9. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    SciTech Connect

    Tsujiuchi, Toshifumi . E-mail: ttujiuch@life.kindai.ac.jp; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-10-27

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.

  10. DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

    PubMed

    Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

    2016-08-01

    Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

  11. Methylthioadenosine phosphorylase gene is silenced by promoter hypermethylation in human lymphoma cell line DHL-9: another mechanism of enzyme deficiency.

    PubMed

    Ishii, Masaaki; Nakazawa, Keiko; Wada, Hideo; Nishioka, Junji; Nakatani, Kaname; Yamada, Yasuaki; Kamihira, Shimeru; Kusunoki, Masato; Nobori, Tsutomu

    2005-04-01

    Methylthioadenosine phosphorylase (MTAP) involved in the metabolism of purine and polyamine has been known to be deficient in a variety of tumors. Although this enzyme deficiency was reportedly caused by partial or total deletion of the MTAP gene, human MTAP-deficient lymphoma cell line DHL-9 has the intact MTAP gene. In order to determine the mechanism of MTAP deficiency in DHL-9, we carried out methylation-specific PCR analysis of sodium bisulfite-treated genomic DNA followed by DNA sequence analysis. Following incubation with various concentrations of 5-Aza-2'-deoxycytidine, DHL-9 cells were subjected to RT-PCR and an immunoblot analysis for MTAP expression. MTAP promoter in DHL-9 cells was methylated at cytosine of all CpG dinucleotides analyzed. Moreover, 5-Aza-2'-deoxycytidine treatment induced DHL-9 cells to express MTAP mRNA and protein. Taken together, MTAP deficiency in DHL-9 was caused by transcriptional silencing due to promoter methylation. Promoter methylation of the MTAP gene was also found in DNA samples from adult T-cell leukemia patients. These results indicated that promoter hypermethylation is another mechanism of MTAP deficiency in human malignancy. Thus, immunological diagnostics will be needed for an accurate evaluation of MTAP expression at the protein level.

  12. Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

    PubMed Central

    Matsushita, Maiko; Otsuka, Yohei; Tsutsumida, Naoya; Tanaka, Chiaki; Uchiumi, Akane; Ozawa, Koji; Suzuki, Takuma; Ichikawa, Daiju; Aburatani, Hiroyuki; Okamoto, Shinichiro; Kawakami, Yutaka; Hattori, Yutaka

    2016-01-01

    The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients. PMID:26784514

  13. The clinical implication of single nucleotide polymorphisms in deoxycytidine kinase in chronic hepatitis B patients treated with lamivudine.

    PubMed

    Lee, Hyun Woong; Lee, Sung Hee; Lee, Min Goo; Ahn, Sang Hoon; Chang, Hye Young; Han, Kwang-Hyub

    2016-05-01

    Deoxycytidine kinase (dCK) is a critical enzyme involved in intracellular phosphorylation of lamivudine (LAM) to its active triphosphates. We conducted this study to determine dCK polymorphisms in Koreans and to evaluate whether the discovered single nucleotide polymorphisms (SNPs) were associated with treatment outcomes in chronic hepatitis B (CHB) patients treated with LAM. The full-length dCK gene was sequenced from 24 healthy volunteers and 24 patients with CHB. One hundred twenty-seven patients with CHB who were followed-up for at least 24 months after LAM treatment were enrolled. Virological response as determined by undetectable HBV DNA was defined as a good drug response. Primary non-response at 6 months and virological breakthrough within 12 months were defined as a poor drug response. Six novel dCK SNPs were found (-2052C/A, IVS3 - 46G/del, IVS4 + 40G/T, IVS5 + 39T/C, IVS5 - 72A/T, and 966-975T10/T11). In particular, two promoter SNPs, namely -360C/G and -201C/T, were in full linkage disequilibrium. These two SNPs had a higher allele frequency than previously reported in Caucasian, Japanese, and Chinese (26% vs. 2%, 13.1%, and 15.6%, respectively). There was no significant difference between treatment response groups in terms of the distributions of SNP genotypes or allele frequencies. However, there was significant difference in the allele frequency of -360G/-201T between HBeAg seroclearance group and HBeAg non-seroclearance group (P = 0.045). In conclusion, six novel dCK SNPs were discovered. Two promoter SNPs, namely -360C/G and -201C/T, were more frequent in Koreans than other populations. In particular, HBeAg-positive patients with the -360G/-201T haplotype may help HBeAg seroclearance in response to LAM therapy. © 2015 Wiley Periodicals, Inc.

  14. Development of new deoxycytidine kinase inhibitors and non-invasive in vivo evaluation using Positron Emission Tomography

    PubMed Central

    Murphy, Jennifer M.; Armijo, Amanda L.; Nomme, Julian; Lee, Chi Hang; Smith, Quentin A.; Li, Zheng; Campbell, Dean O.; Liao, Hsiang-I; Nathanson, David A.; Austin, Wayne R.; Lee, Jason T.; Darvish, Ryan; Wei, Liu; Wang, Jue; Su, Ying; Damoiseaux, Robert; Sadeghi, Saman; Phelps, Michael E.; Herschman, Harvey R.; Czernin, Johannes; Alexandrova, Anastassia N.; Jung, Michael E.; Lavie, Arnon; Radu, Caius G.

    2013-01-01

    Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using 18F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine (18F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1 – 12 nM) using the 18F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy. PMID:23947754

  15. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    NASA Astrophysics Data System (ADS)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  16. Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification.

    PubMed

    Pham, Phuong; Afif, Samir A; Shimoda, Mayuko; Maeda, Kazuhiko; Sakaguchi, Nobuo; Pedersen, Lars C; Goodman, Myron F

    2016-07-01

    Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating C→U during transcription of Ig-variable (V) and Ig-switch (S) region DNA, which is essential to produce high-affinity antibodies. Here we report the crystal structure of a soluble human AID variant at 2.8Å resolution that favors targeting WRC motifs (W=A/T, R=A/G) in vitro, and executes Ig V SHM in Ramos B-cells. A specificity loop extending away from the active site to accommodate two purine bases next to C, differs significantly in sequence, length, and conformation from APOBEC proteins Apo3A and Apo3G, which strongly favor pyrimidines at -1 and -2 positions. Individual amino acid contributions to specificity and processivity were measured in relation to a proposed ssDNA binding cleft. This study provides a structural basis for residue contributions to DNA scanning properties unique to AID, and for disease mutations in human HIGM-2 syndrome. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Antiproliferative effects of sapacitabine (CYC682), a novel 2'-deoxycytidine-derivative, in human cancer cells.

    PubMed

    Serova, M; Galmarini, C M; Ghoul, A; Benhadji, K; Green, S R; Chiao, J; Faivre, S; Cvitkovic, E; Le Tourneau, C; Calvo, F; Raymond, E

    2007-09-03

    This study assessed the antiproliferative activity of sapacitabine (CYC682, CS-682) in a panel of 10 human cancer cell lines with varying degrees of resistance or sensitivity to the commonly used nucleoside analogues ara-C and gemcitabine. Growth inhibition studies using sapacitabine and CNDAC were performed in the panel of cell lines and compared with both nucleoside analogues and other anticancer compounds including oxaliplatin, doxorubicin, docetaxel and seliciclib. Sapacitabine displayed antiproliferative activity across a range of concentrations in a variety of cell lines, including those shown to be resistant to several anticancer drugs. Sapacitabine is biotransformed by plasma, gut and liver amidases into CNDAC and causes cell cycle arrest predominantly in the G(2)/M phase. No clear correlation was observed between sensitivity to sapacitabine and the expression of critical factors involved in resistance to nucleoside analogues such as deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1, cytosolic 5'-nucleotidase and DNA polymerase-alpha. However, sapacitabine showed cytotoxic activity against dCK-deficient L1210 cells indicating that in some cells, a dCK-independent mechanism of action may be involved. In addition, sapacitabine showed a synergistic effect when combined with gemcitabine and sequence-specific synergy with doxorubicin and oxaliplatin. Sapacitabine is therefore a good candidate for further evaluation in combination with currently used anticancer agents in tumour types with unmet needs.

  18. Stereochemical preference of 2'-deoxycytidine for chiral bis(diamido)-bridged basket resorcin[4]arenes.

    PubMed

    D'Acquarica, Ilaria; Calcaterra, Andrea; Sacco, Fabiola; Balzano, Federica; Aiello, Federica; Tafi, Andrea; Pesci, Nicolò; Uccello-Barretta, Gloria; Botta, Bruno

    2013-12-01

    Bis(diamido)-bridged basket resorcin[4]arene (all-S)-1 and its (all-R)-1 enantiomer proved able to interact with 2'-deoxycytidine (2) and pyrimidine nucleoside analogs in dimethyl sulfoxide (DMSO) solution. In such a solvent, the resorcinarene hosts adopt a preferential 1,3-alternate-like conformation, with a larger cavity delimited by two syn 3,5-dimethoxyphenyl moieties, and two external pockets, each delimited by the other 3,5-dimethoxyphenyl group and its diamido arm (the wing). Complexation phenomena were investigated by nuclear magnetic resonance (NMR) methods, including (1)H NMR DOSY and 1D ROESY experiments, and molecular modeling. Heteroassociation constants of [(all-S)-1·2] and [(all-R)-1·2] diastereoisomeric complexes were obtained from diffusion data by single point measurements, and from nonlinear fitting of 1H NMR chemical shifts. Selective proton relaxation rate measurements allowed us to significantly discriminate the two complexes by identifying two different interaction sites of the guest in the resorcin[4]arene host, depending on its configuration.

  19. Evaluation of fluorescent analogs of deoxycytidine for monitoring DNA transitions from duplex to functional structures.

    PubMed

    Bhavsar, Yogini P; Reilly, Samantha M; Wadkins, Randy M

    2011-01-01

    Topological variants of single-strand DNA (ssDNA) structures, referred to as "functional DNA," have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (tC(°)), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC(°) is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  20. Elucidation of Different Binding Modes of Purine Nucleosides to Human Deoxycytidine Kinase

    SciTech Connect

    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Lavie, Arnon

    2008-07-30

    Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylated at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.

  1. Thrombospondin-1 expression in melanoma is blocked by methylation and targeted reversal by 5-Aza-deoxycytidine suppresses angiogenesis.

    PubMed

    Lindner, Daniel J; Wu, Yan; Haney, Rebecca; Jacobs, Barbara S; Fruehauf, John P; Tuthill, Ralph; Borden, Ernest C

    2013-03-11

    Reversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated. 5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of

  2. Role of Genetic Polymorphisms of Deoxycytidine Kinase and Cytidine Deaminase to Predict Risk of Death in Children with Acute Myeloid Leukemia

    PubMed Central

    Medina-Sanson, Aurora; Ramírez-Pacheco, Arturo; Moreno-Guerrero, Silvia Selene; Dorantes-Acosta, Elisa María; Sánchez-Preza, Metzeri; Reyes-López, Alfonso

    2015-01-01

    Cytarabine is one of the most effective antineoplastic agents among those used for the treatment of acute myeloid leukemia. However, some patients develop resistance and/or severe side effects to the drug, which may interfere with the efficacy of the treatment. The polymorphisms of some Ara-C metabolizing enzymes seem to affect outcome and toxicity in AML patients receiving cytarabine. We conducted this study in a cohort of Mexican pediatric patients with AML to investigate whether the polymorphisms of the deoxycytidine kinase and cytidine deaminase enzymes are implicated in clinical response and toxicity. Bone marrow and/or peripheral blood samples obtained at diagnosis from 27 previously untreated pediatric patients with de novo AML were processed for genotyping and in vitro chemosensitivity assay, and we analyzed the impact of genotypes and in vitro sensitivity on disease outcome and toxicity. In the multivariate Cox regression analysis, we found that age at diagnosis, wild-type genotype of the CDA A79C polymorphism, and wild-type genotype of the dCK C360G polymorphism were the most significant prognostic factors for predicting the risk of death. PMID:26090398

  3. Role of Genetic Polymorphisms of Deoxycytidine Kinase and Cytidine Deaminase to Predict Risk of Death in Children with Acute Myeloid Leukemia.

    PubMed

    Medina-Sanson, Aurora; Ramírez-Pacheco, Arturo; Moreno-Guerrero, Silvia Selene; Dorantes-Acosta, Elisa María; Sánchez-Preza, Metzeri; Reyes-López, Alfonso

    2015-01-01

    Cytarabine is one of the most effective antineoplastic agents among those used for the treatment of acute myeloid leukemia. However, some patients develop resistance and/or severe side effects to the drug, which may interfere with the efficacy of the treatment. The polymorphisms of some Ara-C metabolizing enzymes seem to affect outcome and toxicity in AML patients receiving cytarabine. We conducted this study in a cohort of Mexican pediatric patients with AML to investigate whether the polymorphisms of the deoxycytidine kinase and cytidine deaminase enzymes are implicated in clinical response and toxicity. Bone marrow and/or peripheral blood samples obtained at diagnosis from 27 previously untreated pediatric patients with de novo AML were processed for genotyping and in vitro chemosensitivity assay, and we analyzed the impact of genotypes and in vitro sensitivity on disease outcome and toxicity. In the multivariate Cox regression analysis, we found that age at diagnosis, wild-type genotype of the CDA A79C polymorphism, and wild-type genotype of the dCK C360G polymorphism were the most significant prognostic factors for predicting the risk of death.

  4. Origin of increased deoxycytidine excretion into urine of rats bearing Yoshida ascites sarcoma

    SciTech Connect

    Shimizu, M.; Fujimura, S.

    1984-06-01

    The metabolism of deoxycytidine (dCyd) and dCyd nucleotides in Yoshida ascites sarcoma (YS) cells and the host rat liver was investigated with reference to the increased excretion of urinary dCyd. Incorporation of (/sup 14/C)orotic acid into the livers of rats at the fifth day after the transplantation of YS cells, was 2 times higher than that into the normal rat livers. After the injection of (/sup 14/C)orotic acid, the ratio of the specific radioactivity of cytidylate to uridylate moieties of the host liver RNA was measured and found to be higher than that of normal rat liver RNA and to be similar to that of YS cell RNA. When (/sup 14/C)orotic acid was injected into rats followed by the transplantation of YS cells, the radioactivities present in the livers disappeared more rapidly than those in the control rat livers. The activities of pyrimidine de novo synthesis enzymes, such as cytidine triphosphate synthetase and cytidine diphosphate reductase, in YS were higher than those in both rat ascites hepatoma AH 7974 and Walker 256 carcinosarcoma, the transplantations of which did not induce increased excretion of dCyd into urine of the hosts. The activities of dCyd kinase and dCyd deaminase in YS cells were lower than those in the other two tumors investigated. The activities of cytidine triphosphate synthetase and cytidine diphosphate reductase in the livers of YS-bearing rats were elevated compared with those in the livers of rat ascites hepatoma AH 7974- or Walker 256 carcinosarcoma-bearing rats and normal rats, while the activities of dCyd kinase, 5'-nucleotidase, and dCyd deaminase were similar between normal rat livers and tumor-bearing rat livers.

  5. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    SciTech Connect

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  6. Activation of an imprinted Igf 2 gene in mouse somatic cell cultures.

    PubMed Central

    Eversole-Cire, P; Ferguson-Smith, A C; Sasaki, H; Brown, K D; Cattanach, B M; Gonzales, F A; Surani, M A; Jones, P A

    1993-01-01

    The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine. Images PMID:8336727

  7. Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5'-nucleotidase I activity in MOLT-4 cells.

    PubMed

    Fyrberg, A; Mirzaee, S; Lotfi, K

    2006-01-01

    Activation of nucleoside analogues is dependent on kinases and 5'-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5'-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5'-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.

  8. Electron attachment to a hydrated DNA duplex: the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine.

    PubMed

    Gu, Jiande; Wong, Ning-Bew; Xie, Yaoming; Schaefer, F Henry

    2010-11-22

    The minimal essential section of DNA helices, the dinucleoside phosphate deoxyguanylyl-3',5'-deoxycytidine dimer octahydrate, [dGpdC](2), has been constructed, fully optimized, and analyzed by using quantum chemical methods at the B3LYP/6-31+G(d,p) level of theory. Study of the electrons attached to [dGpdC](2) reveals that DNA double strands are capable of capturing low-energy electrons and forming electronically stable radical anions. The relatively large vertical electron affinity (VEA) predicted for [dGpdC](2) (0.38 eV) indicates that the cytosine bases are good electron captors in DNA double strands. The structure, charge distribution, and molecular orbital analysis for the fully optimized radical anion [dGpdC](2)(·-) suggest that the extra electron tends to be redistributed to one of the cytosine base moieties, in an electronically stable structure (with adiabatic electron affinity (AEA) 1.14 eV and vertical detachment energy (VDE) 2.20 eV). The structural features of the optimized radical anion [dGpdC](2)(·-) also suggest the probability of interstrand proton transfer. The interstrand proton transfer leads to a distonic radical anion [d(G-H)pdC:d(C+H)pdG](·-), which contains one deprotonated guanine anion and one protonated cytosine radical. This distonic radical anion is predicted to be more stable than [dGpdC](2)(·-). Therefore, experimental evidence for electron attachment to the DNA double helices should be related to [d(G-H)pdC:d(C+H)pdG](·-) complexes, for which the VDE might be as high as 2.7 eV (in dry conditions) to 3.3 eV (in fully hydrated conditions). Effects of the polarizable medium have been found to be important for increasing the electron capture ability of the dGpdC dimer. The ultimate AEA value for cytosine in DNA duplexes is predicted to be 2.03 eV in aqueous solution.

  9. Determination of 5-methyl-2'-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection.

    PubMed

    Sandhu, Jatinderpal; Kaur, Balvinder; Armstrong, Christine; Talbot, Christopher J; Steward, William P; Farmer, Peter B; Singh, Rajinder

    2009-07-01

    The formation of 5-methyl-2'-deoxycytidine (5-MedC) following methylation of the C-5 position of cytosine in genomic DNA provides an epigenetic mechanism for the regulation of gene expression and cellular differentiation. We describe the development of a method using HPLC-ultraviolet (UV) detection for the accurate determination of 5-MedC in DNA. Genomic DNA was obtained from HeLa cells and rat liver tissue using an optimised anion-exchange column DNA extraction procedure incorporating a ribonuclease incubation step to remove any potential interference from RNA. Following extraction the DNA samples were enzymatically hydrolysed to 2'-deoxynucleosides using a combination of an endo-exonuclease plus 5'-exonuclease together with a 3'-nucleotidase. The hydrolysed DNA samples (10 microg on column) were analysed using narrow-bore reverse phase HPLC-UV detection. The level of 5-MedC in the DNA samples was expressed as a percentage of the level of 2'-deoxycytidine (dC) determined from calibration lines constructed using authentic standards for 5-MedC and dC. The percentage 5-MedC level determined for commercially available calf thymus DNA was 6.26%, for HeLa cell DNA was 3.02% and for rat liver DNA was 3.55%.

  10. In silico kinetics and mechanism of interaction of cis-2-butene-1,4-dial with 2'-deoxycytidine.

    PubMed

    Sviatenko, Liudmyla K; Gorb, Leonid; Hovorun, Dmytro; Leszczynski, Jerzy

    2014-06-16

    Newly proposed approach involving computational analysis of multistep chemical reactions has been successfully applied to study the interaction between 2'-deoxycytidine and cis-2-butene-1,4-dial, a metabolite of furan. The new method comprises a combination of few steps. They include the prediction of the reaction mechanism, calculation of Gibbs free energies for the reaction pathway, and conversion of barrier energies to rate constants. On the basis of the results of previous steps, corresponding kinetic equations are constructed and solved. Such a procedure allows one to indicate the definite concentration of reaction species (reactants, intermediates, and products) at any moment in time. Obtained results show that 2'-deoxycytidine reacts with cis-2-butene-1,4-dial to form primary products, which are represented by four polycyclic diastereomers. These primary products further transform to more stable secondary product by dehydration, which is catalyzed by acid. The obtained data demonstrate that cis-2-butene-1,4-dial plays a key role in furan-induced carcinogenesis.

  11. Low energy electron attachment to the nucleotide deoxycytidine monophosphate: direct evidence for the molecular mechanisms of electron-induced DNA strand breaks.

    PubMed

    Kopyra, Janina

    2012-06-21

    Reactions induced by the attachment of low energy electrons to an entire gas phase nucleotide (2'-deoxycytidine 5'-monophosphate) are reported for the first time. From the resonant attachment profiles information on the site of initial electron localization and from the observed ionic fragments information on final bond cleavage can be extracted.

  12. Elucidation of Chromatin Remodeling Machinery Involved in Regulation of Estrogen Receptor Alpha Expression in Human Breast Cancer Cells

    DTIC Science & Technology

    2005-08-01

    5-aza-2’-deoxycytidine. Cancer Res. 62:6456-6461 35. Widschwendter M, Siegmund KD, Muller HM, Fiegl H, Marth C, Muller- Holzner E, Jones PA, and Laird...his- Marth C, Muller- Holzner E, Jones PA, Laird PW 2004 tone H3 methyltransferases. Nature 406:593-599 Association of breast cancer DNA methylation

  13. Potent methyl oxidation of 5-methyl-2'-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism.

    PubMed

    Shao, Jie; Huang, Chun-Hua; Kalyanaraman, Balaraman; Zhu, Ben-Zhan

    2013-07-01

    Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical ((•)OH) can be produced by halogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2'-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2'-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H2O2. The formation of the oxidation products was markedly inhibited by typical (•)OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H2O2 might be through the following mechanism: (•)OH produced by TCBQ/H2O2 may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2'-deoxycytidylyl)methyl radical, which may combine with O2 to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2'-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H2O2 can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensis.

    PubMed

    Firmino, Joana; Carballo, Carlos; Armesto, Paula; Campinho, Marco A; Power, Deborah M; Manchado, Manuel

    2017-07-17

    The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2'-deoxycytidine) and temperature was evaluated. Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2'-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C. Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2'-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in

  15. Investigation of dissociative electron attachment to 2'-deoxycytidine-3'-monophosphate using DFT method and time dependent wave packet approach

    NASA Astrophysics Data System (ADS)

    Bhowmick, Somnath; B, Renjith; Mishra, Manoj K.; Sarma, Manabendra

    2012-08-01

    Effect of electron correlation on single strand breaks (SSBs) induced by low energy electron (LEE) has been investigated in a fragment excised from a DNA, viz., 2'-deoxycytidine-3'-monophosphate [3'-dCMPH] molecule in gas phase at DFT-B3LYP/6-31+G(d) accuracy level and using local complex potential based time dependent wave packet (LCP-TDWP) approach. The results obtained, in conjunction with our earlier investigation, show the possibility of SSB at very low energy (0.15 eV) where the LEE transfers from π* to σ* resonance state which resembles a SN2 type mechanism. In addition, for the first time, an indication of quantum mechanical tunneling in strand breaking is seen from the highest anionic bound vibrational state (χ5), which may have a substantial role during DNA damage.

  16. Reactions of an osmium-hexahydride complex with cytosine, deoxycytidine, and cytidine: the importance of the minor tautomers.

    PubMed

    Esteruelas, Miguel A; García-Raboso, Jorge; Oliván, Montserrat

    2012-09-03

    Complex OsH(6)(P(i)Pr(3))(2) (1) deprotonates cytosine to give molecular hydrogen and the d(4)-trihydride derivative OsH(3)(cytosinate)(P(i)Pr(3))(2) (2), which in solution exists as a mixture of isomers containing κ(2)-N1,O (2a) and κ(2)-N3,O (2b) amino-oxo and κ(2)-N3,N4 (2c) imino-oxo tautomers. The major isomer 2b associates with the minor one 2c through N-H···N and N-H···O hydrogen bonds to form [2b·2c](2) dimers, which crystallize from saturated pentane solutions of 2. Complex 1 is also able to perform the double deprotonation of cytosine (cytosinate') to afford the dinuclear derivative (P(i)Pr(3))(2)H(3)Os(cytosinate')OsH(3)(P(i)Pr(3))(2) (3), where the anion is coordinated κ(2)-N1,O and κ(2)-N3,N4 to two different OsH(3)(P(i)Pr(3))(2) metal fragments. The deprotonation of deoxycytidine and cytidine leads to OsH(3)(deoxycytidinate)(P(i)Pr(3))(2) (4) and OsH(3)(cytidinate)(P(i)Pr(3))(2) (5), respectively, containing the anion κ(2)-N3,N4 coordinated. Dimer [2b·2c](2) and dinuclear complex 3 have been characterized by X-ray diffraction analysis.

  17. Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis

    SciTech Connect

    Chuang, Jing-Jing; Dai, Yuan-Chang; Lin, Yung-Lun; Chen, Yang-Yi; Lin, Wei-Han; Chan, Hong-Lin; Liu, Yi-Wen

    2014-09-15

    Bladder cancer is highly recurrent following specific transurethral resection and intravesical chemotherapy, which has prompted continuing efforts to develop novel therapeutic agents and early-stage diagnostic tools. Specific changes in protein expression can provide a diagnostic marker. In our present study, we investigated changes in protein expression during urothelial carcinogenesis. The carcinogen BBN was used to induce mouse bladder tumor formation. Mouse bladder mucosa proteins were collected and analyzed by 2D electrophoresis from 6 to 20 weeks after commencing continuous BBN treatment. By histological examination, the connective layer of the submucosa showed gradual thickening and the number of submucosal capillaries gradually increased after BBN treatment. At 12-weeks after the start of BBN treatment, the urothelia became moderately dysplastic and tumors arose after 20-weeks of treatment. These induced bladder lesions included carcinoma in situ and connective tissue invasive cancer. In protein 2D analysis, the sequentially downregulated proteins from 6 to 20 weeks included GSTM1, L-lactate dehydrogenase B chain, keratin 8, keratin 18 and major urinary proteins 2 and 11/8. In contrast, the sequentially upregulated proteins identified were GSTO1, keratin 15 and myosin light polypeptide 6. Western blotting confirmed that GSTM1 and NQO-1 were decreased, while GSTO1 and Sp1 were increased, after BBN treatment. In human bladder cancer cells, 5-aza-2′-deoxycytidine increased the GSTM1 mRNA and protein expression. These data suggest that the downregulation of GSTM1 in the urothelia is a biomarker of bladder carcinogenesis and that this may be mediated by DNA CpG methylation. - Highlights: • GSTM1 and NQO-1 proteins decreased in the mouse bladder mucosa after BBN treatment. • BBN induced GSTO1 and Sp1 protein expression in the mouse bladder mucosa. • 5-Aza-2′-deoxycytidine increased GSTM1 mRNA and protein in human bladder cancer cell. • GSTM1

  18. Repression of DOK7 mediated by DNMT3A promotes the proliferation and invasion of KYSE410 and TE-12 ESCC cells.

    PubMed

    Yang, Shou-Mei; Li, Su-Yi; Yu, Hao-Bin; Li, Jie-Ru; Sun, Lei-Lei

    2017-03-23

    Increasing evidence shows that aberrant epigenetic regulation of tumor suppressor genes is a contributing factor to their altered expression in esophageal squamous cell carcinoma (ESCC). In the current study, we investigate the role of DOK7 in ESCC cells. We found that enforced expression of DOK7 inhibited the proliferation and invasion of ESCC cells. We also found that treatment of ESCC cells with the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-azadC), induced the demethylation of DOK7 in promoter and DOK7 expression. Moreover, silencing DNMT3A decreased methylation of DOK7 and increased DOK7 expression, followed by repressing the proliferation and invasion of ESCC cells. Collectively, our data indicated that silencing DNMT3A inhibits proliferation and invasion in ESCC cells by inducing demethylation of DOK7.

  19. Emerging role of N-myc downstream-regulated gene 2 (NDRG2) in cancer

    PubMed Central

    Ma, Zhiqiang; Yan, Xiaolong; Di, Shouyin; Jiang, Shuai; Li, Tian; Cheng, Yedong; Yang, Yang

    2016-01-01

    N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor and cell stress-related gene. NDRG2 is associated with tumor incidence, progression, and metastasis. NDRG2 regulates tumor-associated genes and is regulated by multiple conditions, treatments, and protein/RNA entities, including hyperthermia, trichostatin A and 5-aza-2′-deoxycytidine, which are promising potential cancer therapeutics. In this review, we discuss the expression as well as the clinical and pathological significance of NDRG2 in cancer. The pathological processes and molecular pathways regulated by NDRG2 are also summarized. Moreover, mechanisms for increasing NDRG2 expression in tumors and the potential directions of future NDRG2 research are discussed. The information reviewed here should assist in experimental design and increase the potential of NDRG2 as a therapeutic target for cancer. PMID:26506239

  20. Identification of a novel human cancer/testis gene MAEL that is regulated by DNA methylation.

    PubMed

    Xiao, Ling; Wang, Yijun; Zhou, Yankai; Sun, Yi; Sun, Wei; Wang, Lin; Zhou, Chang; Zhou, Jianlin; Zhang, Jian

    2010-06-01

    The mouse maelstrom (MAEL) gene has been found to be expressed in male germ cells and to play a role in spermatogenesis. Here, we cloned the human MAEL gene by digital differential display and found that, among human tissues, MAEL is only expressed in the testis, but it is also expressed in various cancer cell lines. The transcription start site of the MAEL gene is 74-bp upstream of the start codon. The region from -216 to +150 is the basal promoter of the MAEL gene, and a CpG island (-295 to +148) is located in this region. Treatment with the demethylating agent 5'-Aza-2-Deoxycytidine significantly upregulated MAEL expression. These results suggest that MAEL is a novel cancer/testis-associated gene and is regulated by DNA methylation.

  1. Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome

    PubMed Central

    Tripathi, Satyendra C.; Peters, Haley L.; Taguchi, Ayumu; Katayama, Hiroyuki; Wang, Hong; Momin, Amin; Jolly, Mohit Kumar; Celiktas, Muge; Rodriguez-Canales, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Ben-Jacob, Eshel; Levine, Herbert; Molldrem, Jeffrey J.; Hanash, Samir M.; Ostrin, Edwin J.

    2016-01-01

    The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides. PMID:26929325

  2. Oxidized low-density lipoprotein inhibits THP-1-derived macrophage autophagy via TET2 down-regulation.

    PubMed

    Li, Guohua; Peng, Juan; Liu, Yanhui; Li, Xiaohong; Yang, Qin; Li, Yongqing; Tang, Zhihan; Wang, Zuo; Jiang, Zhisheng; Wei, Dangheng

    2015-02-01

    Oxidized low-density lipoprotein (ox-LDL) is an independent risk factor of atherosclerosis. However, the mechanism underlying its pro-atherosclerosis roles has not yet been well explored. DNA demethylation modification, via DNA methyltransferases or ten-eleven-translocation (TET) family, is a crisis epigenetic regulation for various biological and pathological processes. This study aimed to investigate the effects of ox-LDL on macrophage autophagy and its potential epigenetic mechanism. Results showed that after treatment with 0, 10, 20, 40 or 80 mg/L ox-LDL for 24 h, the autophagy markers Beclin 1 and LC3 expression were obviously decreased at protein levels (P < 0.05). The mRNA and protein expression of TET2 was evidently decreased (P < 0.05). After pre-treatment with TET2 siRNA, the mRNA and protein levels of Beclin 1 and LC3 decreased compared with the 80 mg/L treatment group (P < 0.01). The mRNA and protein levels of Beclin 1 and LC3-II were up-regulated (P < 0.05) in the 5-aza-2'-deoxycytidine (a DNA methyltransferase inhibitor) of pretreatment group. Consistent with the Western blot results, cell immunofluorescence showed that the protein concentration of LC3-II decreased in the TET2 siRNA group and increased in the 5-aza-2'-deoxycytidine group. Taken together, these results showed that DNA demethylation modifications regulate ox-LDL-treated THP-1 macrophages autophagy and TET2 might be a novel regulator.

  3. Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma.

    PubMed

    Hirata, Hiroshi; Hinoda, Yuji; Nakajima, Koichi; Kawamoto, Ken; Kikuno, Nobuyuki; Ueno, Koji; Yamamura, Soichiro; Zaman, Mohd S; Khatri, Gaurav; Chen, Yi; Saini, Sharanjot; Majid, Shahana; Deng, Guoren; Ishii, Nobuhisa; Dahiya, Rajvir

    2011-04-15

    The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2'-deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.

  4. Upregulated INHBA Expression May Promote Cell Proliferation and Is Associated with Poor Survival in Lung Adenocarcinoma1

    PubMed Central

    Seder, Christopher W; Hartojo, Wibisono; Lin, Lin; Silvers, Amy L; Wang, Zhuwen; Thomas, Dafydd G; Giordano, Thomas J; Chen, Guoan; Chang, Andrew C; Orringer, Mark B; Beer, David G

    2009-01-01

    Introduction The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated. Methods INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression. Results Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin βA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2deoxycytidine. Conclusions INHBA is overexpressed in AD relative to controls. Inhibin βA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs. PMID:19308293

  5. Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

    PubMed

    Chan, Michael W Y; Wei, Susan H; Wen, Ping; Wang, Zailong; Matei, Daniela E; Liu, Joseph C; Liyanarachchi, Sandya; Brown, Robert; Nephew, Kenneth P; Yan, Pearlly S; Huang, Tim H-M

    2005-10-15

    Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.

  6. Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

    PubMed

    Rius, Maria; Stresemann, Carlo; Keller, Daniela; Brom, Manuela; Schirrmacher, Esther; Keppler, Dietrich; Lyko, Frank

    2009-01-01

    The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

  7. Genome-wide unmasking of epigenetically silenced genes in lung adenocarcinoma from smokers and never smokers

    PubMed Central

    Yingling, Christin M.; Liu, Yushi; Tellez, Carmen S.; Van Neste, Leander; Baylin, Stephen S.; Belinsky, Steven A.

    2014-01-01

    Lung cancer in never smokers (NS) shows striking demographic, clinicopathological and molecular distinctions from the disease in smokers (S). Studies on selected genetic and epigenetic alterations in lung cancer identified that the frequency and profile of some abnormalities significantly differ by smoking status. This study compared the transcriptome of lung adenocarcinoma cell lines derived from S (n = 3) and NS (n = 3) each treated with vehicle (control), histone deacetylation inhibitor (trichostatin A) or DNA methylation inhibitor (5-aza-2′-deoxycytidine). Among 122 genes reexpressed following 5-aza-2′-deoxycytidine but not trichostatin A treatment in two or more cell lines (including 32 genes in S-only and 12 NS-only), methylation was validated for 80% (98/122 genes). After methylation analysis of 20 normal tissue samples and 14 additional non–small cell lung cancer cell lines (total 20), 39 genes frequently methylated in normal (>20%, 4/20) and 21 genes rarely methylated in non–small cell lung cancer (≤10%, 2/20) were excluded. The prevalence for methylation of the remaining 38 genes in lung adenocarcinomas from S (n = 97) and NS (n = 75) ranged from 8–89% and significantly differs between S and NS for CPEB1, CST6, EMILIN2, LAYN and MARVELD3 (P < 0.05). Furthermore, methylation of EMILIN2, ROBO3 and IGDCC4 was more prevalent in advanced (Stage II–IV, n = 61) than early (Stage I, n = 110) tumors. Knockdown of MARVELD3, one of the novel epigenetically silenced genes, by small interfering RNA significantly reduced anchorage-independent growth of lung cancer cells (P < 0.001). Collectively, this study has identified multiple, novel, epigenetically silenced genes in lung cancer and provides invaluable resources for the development of diagnostic and prognostic biomarkers. PMID:24398667

  8. Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors

    PubMed Central

    Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

    2016-01-01

    Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation—Pdx1, Neurogenin3, and MafA—efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2′-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2′-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy. PMID:27187823

  9. PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

    PubMed

    Flores-Juárez, Cyntia R; González-Jasso, Eva; Antaramian, Anaid; Pless, Reynaldo C

    2016-10-01

    GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

  10. Structural basis for activation of the therapeutic l-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase

    PubMed Central

    Sabini, Elisabetta; Hazra, Saugata; Konrad, Manfred; Burley, Stephen K.; Lavie, Arnon

    2007-01-01

    l-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the l-nucleosides (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC)—an approved anti-human immunodeficiency virus (HIV) agent—and troxacitabine (TRO)—an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural d-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both d- and l-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the l- and d-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of l-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class. PMID:17158155

  11. A phase I, pharmacokinetic, and pharmacodynamic evaluation of the DNA methyltransferase inhibitor 5-fuoro-2′-deoxycytidine, administered with tetrahydrouridine

    PubMed Central

    Morgan, Robert J.; Kummar, Shivaani; Beumer, Jan H.; Blanchard, M. Suzette; Ruel, Christopher; El-Khoueiry, Anthony B.; Carroll, Mary I.; Hou, Jessie M.; Li, Chun; Lenz, Heinz J.; Eiseman, Julie L.; Doroshow, James H.

    2015-01-01

    Purpose Inhibitors of DNA (cytosine-5)-methyltransferases (DNMT) are active antineoplastic agents. We conducted the first-in-human phase I trial of 5-fluoro-2′-deoxycytidine (FdCyd), a DNMT inhibitor stable in aqueous solution, in patients with advanced solid tumors. Objectives were to establish the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of FdCyd + tetrahydrouridine (THU). Methods FdCyd + THU were administered by 3 h IV infusion on days 1–5 every 3 weeks, or days 1–5 and 8–12 every 4 weeks. FdCyd was administered IV with a fixed 350 mg/m2/day dose of THU to inhibit deamination of FdCyd. Pharmacokinetics of FdCyd, downstream metabolites and THU were assessed by LC–MS/MS. RBC γ-globin expression was evaluated as a pharmacodynamics biomarker. Results Patients were enrolled on the 3-week schedule at doses up to 80 mg/m2/day without dose-limiting toxicity (DLT) prior to transitioning to the 4-week schedule, which resulted in an MTD of 134 mg/m2/day; one of six patients had a first-cycle DLT (grade 3 colitis). FdCyd ≥40 mg/m2/day produced peak plasma concentrations >1 μM. Although there was inter-patient variability, γ-globin mRNA increased during the first two treatment cycles. One refractory breast cancer patient experienced a partial response (PR) of >90 % decrease in tumor size, lasting over a year. Conclusions The MTD was established at 134 mg/m2 FdCyd + 350 mg/m2 THU days 1–5 and 8–12 every 4 weeks. Based on toxicities observed over multiple cycles, good plasma exposures, and the sustained PR observed at 67 mg/m2/day, the phase II dose for our ongoing multi-histology trial is 100 mg/m2/day FdCyd with 350 mg/m2/day THU. PMID:25567350

  12. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko

    2010-01-01

    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  13. Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine

    PubMed Central

    Kay, Brian K.; Lavie, Arnon

    2016-01-01

    Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells. PMID:27280468

  14. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine.

    PubMed

    Ligasová, Anna; Liboska, Radek; Friedecký, David; Mičová, Kateřina; Adam, Tomáš; Oždian, Tomáš; Rosenberg, Ivan; Koberna, Karel

    2016-01-01

    5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA. © 2016 The Authors.

  15. Regulation of deoxycytidine kinase expression and sensitivity to gemcitabine by micro-RNA 330 and promoter methylation in cancer cells.

    PubMed

    Hodzic, Jasmina; Giovannetti, Elisa; Diosdado, Begoňa; Calvo, Begona Diosdado; Adema, A D; Peters, G J

    2011-12-01

    Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation. We hypothesized that dCK could be regulated by two additional mechanisms: micro-RNA (miRNA) and promoter methylation. Methylation-specific PCR (MSP) revealed methylation of the 3' GC box in three out of six cancer cell lines. The 3' GC box is located at the dCK promoter region. The methylation status was related to dCK mRNA expression. TargetScan and miRanda prediction algorithms revealed several possible miRNAs targeting dCK and identified miR-330 (micro-RNA 330) as the one conserved between the human, the chimpanzee, and the rhesus monkey genomes. Expression of miR-330 in various colon and lung cancer cell lines, as measured by QRT-PCR, varied five-fold between samples and correlated with in-vitro gemcitabine resistance (R = 0.82, p = 0.04). Exposure to gemcitabine also appeared to influence miR-330 levels in these cell lines. Furthermore, in our cell line panel, miR-330 expression negatively correlated with dCK mRNA expression (R = 0.74), suggesting a role of miR-330 in post-transcriptional regulation of dCK. In conclusion, the 3' GC box and miR-330 may regulate dCK expression in cancer cells.

  16. Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA.

    PubMed

    Watson, W P; Aston, J P; Barlow, T; Crane, A E; Potter, D; Brown, T

    1999-01-01

    1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the

  17. A density functional theory study on the kinetics and thermodynamics of N-glycosidic bond cleavage in 5-substituted 2'-deoxycytidines.

    PubMed

    Williams, Renee T; Wang, Yinsheng

    2012-08-14

    B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) density functional theory calculations were employed to explore the kinetics and thermodynamics of gas-phase N-glycosidic bond cleavage induced by nucleophilic attack of C1' with a hydroxide ion in 5-substituted 2'-deoxycytidines. The results showed that, among the 5-substituted 2'-deoxycytidine derivatives examined [XdC, where X = H (dC), CH(3) (medC), CH(2)OH (hmdC), CHO (fmdC), COOH (cadC), F (FdC), or Br (BrdC)], fmdC and cadC exhibited the lowest energy barrier and largest exothermicity for N-glycosidic bond cleavage. These results paralleled previously reported nucleobase excision activities of human thymine DNA glycosylase (hTDG) toward duplex DNA substrates harboring a thymine and 5-substituted cytosine derivatives when paired with a guanine. Our study suggests that the inherent chemistry associated with the nucleophilic cleavage of N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG toward 5-substituted dC derivatives. These findings provided novel insights into the role of TDG in active cytosine demethylation.

  18. High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

    PubMed

    Fraga, Mario F; Uriol, Esther; Borja Diego, L; Berdasco, María; Esteller, Manel; Cañal, María Jesús; Rodríguez, Roberto

    2002-06-01

    A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

  19. An oligodeoxyribonucleotide containing 5-formyl-2'-deoxycytidine (fC) at the CpG site forms a covalent complex with DNA cytosine-5 methyltransferases (DNMTs).

    PubMed

    Sato, Kousuke; Kawamoto, Kyoji; Shimamura, Shintaro; Ichikawa, Satoshi; Matsuda, Akira

    2016-11-15

    5-Methylcytosine (mC) is known to induce epigenetic changes. Ten-eleven translocation (TET) enzymes produce the further oxidized 5-substituted cytosine derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC). However, their roles are unclear thus far. Here, we synthesized oligodeoxyribonucleotides (ODNs) containing 5-formyl-2'-deoxycytidine and examined their interactions with DNA cytosine-5 methyltransferase (DNMT). We found that the ODN sequence containing fCpG formed a covalent complex with both bacterial and mouse recombinant DNMTs in the absence of any cofactors. The covalent bonding with DNMT suggests that the fCpG sequence in DNA may play a role in epigenetic regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Fused-core silica column ultra performance liquid chromatography – ion trap tandem mass spectrometry for determination of global DNA methylation status1

    PubMed Central

    Yang, Ill; Fortin, Marie C.; Richardson, Jason R.; Buckley, Brian

    2010-01-01

    Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2′-deoxyguanosine (2dG) and 5-methyl-2′-deoxycytidine (5mdC) were resolved in less than 1 minute, with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG respectively. The accuracy of detection was 95% or above and the day-to-day coefficient of variations was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large scale analysis of epidemiological or clinical DNA samples. PMID:20950581

  1. Gemcitabine, Pyrrologemcitabine and 2'-Fluoro- 2'-Deoxycytidines: Synthesis, Physical Properties and Impact of Sugar Fluorination on Silver Ion Mediated Base Pairing.

    PubMed

    Seela, Frank; Guo, Xiurong; Leonard, Peter; Ingale, Sachin Asaram

    2017-09-14

    The stability of silver mediated "dC-dC" base pairs relies not only on the structure of the nucleobase but also is sensitive to structural modification on the sugar moiety. 2'-Fluorinated 2'-deoxycytidines with fluorine atoms in the arabino (up) and the ribo (down) configuration, as well as with geminal fluorine substitution (anticancer drug gemcitabine) and the novel fluorescent phenylpyrrolo-gemcitabine (phPyrGem) were synthesized. All nucleosides display the recognition face of naturally occurring 2'-deoxycytidine. Nucleosides were converted into phosphoramidites and incorporated in 12-mer oligonucleotides by solid-phase synthesis. Addition of silver ions to DNA duplexes with a fluorine modified "dC-dC" pair near central position led to significant duplex stabilization. The stability increase was higher for duplexes with fluorinated sugar residues than those with the unchanged 2'-deoxyribose moiety. Similar observations were made on "dC-dT" pairs and to a minor extend on the "dC-dA" pairs. The increase of silver ion mediated base pair stability was reversed by annulation of a pyrrole ring to the cytosine moiety as shown for 2'-fluorinated phPyrGem compared to phenylpyrrolo-dC (phPyrdC). The phenomenon results from stereoelectronic effects induced by fluoro substitution which are transmitted from the sugar moiety to the silver ion mediated base pairs. This depends on the number of fluorine substituents, their configuration and the structure of the nucleobase. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Epigenetic Silencing and Resistance to Imatinib Mesylate in CML

    DTIC Science & Technology

    2005-07-01

    of Leukemia .... and Experimental Therapeutics. A 6 6 T A C M D. Anderson Cancer Center, Houston. TX. Purpose To determine the activity of...Jaenisch R: Toxicity of genetic responses in patients with accelerated chronic myeloid leukemia . Cancer Cell 2:117- 5-aza-2’-deoxycytidine to mammalian...with chronic myeloge- 5. Leone G, Voso MT, Teofili L, at at: Inhib- to imatinib mesylate in chronic myelogenous nous leukemia . Cancer 98:522-528, 2003

  3. Reversin increase the plasticity of bone marrow-derived mesenchymal stem cell for generation of cardiomyocyte in vitro.

    PubMed

    Pikir, Budi S; Susilowati, Helen; Hendrianto, Eryk; Abdulrantam, Fedik

    2012-01-01

    to speed up transdifferentiation of bone marrow-derived stem cells into cardiomyocyte in vitro by inducing dedifferentiation of bone marrow-derived mesenchymal stem cell, before induction by 5-aza-2'-deoxycytidine into cardiomyocyte. two-three months old 2.5 kg weight adult male New Zealanad Rabbits were anesthezied with ether, thigh bones were excised, and bone marrow cells were obtained by aspiration. in our experiments after 1 week of mesenchymal stem cell cultures, 20 nM reversin was given to induce dediferentiation and after 24 hours exposure with 9μM 5-aza-2-deoxycytidine, early phase of cardiomyocyte differentiation appeared as cultured cell were strongly positive for GATA-4 and weakly positive for MLC-2ά, although beating cardiomyocyte has not yet appeared at the end of experiment. These experiments also showed a marked CD34+ and c-kit+ gene expression on RT-PCR examination. reversine increase plasticity of bone marrow-derived mesenchymal stem cell to generate cardiomyocyte after 5-aza-2'-deoxycytidine induction. CD34+ and c-kit+ may be a marker for cardiomyocyte progenitor cells.

  4. Characterization of an adenosine deaminase-deficient human histiocytic lymphoma cell line (DHL-9) and selection of mutants deficient in adenosir kinase and deoxycytidine kinase.

    PubMed

    Kubota, M; Kamatani, N; Daddona, P E; Carson, D A

    1983-06-01

    The association of adenosine deaminase (ADA) deficiency with immunodeficiency disease has emphasized the importance of this purine metabolic enzyme for human lymphocyte growth and function. This report describes the natural occurrence of ADA deficiency in a human histiocytic lymphoma cell line, DHL-9. The minimal ADA activity in DHL-9 extracts, 0.028 nmol/min/mg protein, was less than 50% of the activity in two B-lymphoblastoid cell lines from ADA-deficient patients and was resistant to the potent ADA inhibitor deoxycoformycin. A sensitive radioimmunoassay failed to detect immunoreactive ADA in DHL-9 cells. Moreover, in DHL-9 cells, deoxycoformycin did not augment either the growth-inhibitory effects of adenosine and deoxyadenosine or the accumulation of deoxyadenosine triphosphate from deoxyadenosine. When compared to six other human hematopoietic cell lines, DHL-9 had 5.6-fold-higher levels of adenosylhomocysteinase. Chromosome 20, which bears the structural gene for ADA and adenosylhomocysteinase, was diploid and had a normal Giemsa banding pattern. The parental DHL-9 cell line was used for the selection and cloning of secondary mutants deficient in deoxycytidine kinase and adenosine kinase.

  5. Low energy electron induced cytosine base release in 2′-deoxycytidine-3′-monophosphate via glycosidic bond cleavage: A time-dependent wavepacket study

    SciTech Connect

    Bhaskaran, Renjith; Sarma, Manabendra

    2014-09-14

    Low energy electron (LEE) induced cytosine base release in a selected pyrimidine nucleotide, viz., 2′-deoxycytidine-3′-monophosphate is investigated using ab initio electronic structure methods and time dependent quantum mechanical calculations. It has been noted that the cytosine base scission is comparatively difficult process than the 3′ C–O bond cleavage from the lowest π{sup *} shape resonance in energy region <1 eV. This is mainly due to the high activation energy barrier associated with the electron transfer from the π{sup *} orbital of the base to the σ{sup *} orbital of the glycosidic N–C bond. In addition, the metastable state formed after impinging LEE (0–1 eV) has very short lifetime (10 fs) which may decay in either of the two competing auto-detachment or dissociation process simultaneously. On the other hand, the selected N–C mode may cleave to form the cytosine base anion at higher energy regions (>2 eV) via tunneling of the glycosidic bond. Resonance states generated within this energy regime will exist for a duration of ∼35–55 fs. Comparison of salient features of the two dissociation events, i.e., 3′ C–O single strand break and glycosidic N–C bond cleavage in 3′-dCMPH molecule are also provided.

  6. Trichoderma harzianum ETS 323-mediated resistance in Brassica oleracea var. capitata to Rhizoctonia solani involves the novel expression of a glutathione S-transferase and a deoxycytidine deaminase.

    PubMed

    Shibu, Marthandam Asokan; Lin, Hong-Shin; Yang, Hsueh-Hui; Peng, Kou-Cheng

    2012-10-31

    Plant interactions with microbial biocontrol agents are used as experimental models to understand resistance-related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic "programmed cell death" that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H(2)O(2)-inducible glutathione S-transferase (BoGST), which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by reverse-transcription polymerase chain reaction analysis and were found to be transcriptionally activated during the three-way interaction.

  7. Irmpd Action Spectroscopy and Computational Approaches to Elucidate Gas-Phase Structures and Energetics of 2'-DEOXYCYTIDINE and Cytidine Sodium Complexes

    NASA Astrophysics Data System (ADS)

    Zhu, Yanlong; Hamlow, Lucas; He, Chenchen; Gao, Juehan; Oomens, Jos; Rodgers, M. T.

    2016-06-01

    The local structures of DNA and RNA are influenced by protonation, deprotonation and noncovalent interactions with cations. In order to determine the effects of Na+ cationization on the gas-phase structures of 2'-deoxycytidine, [dCyd+Na]+, and cytidine, [Cyd+Na]+, infrared multiple photon dissociation (IRMPD) action spectra of these sodium cationized nucleosides are measured over the range extending from 500 to 1850 wn using the FELIX free electron laser. Complementary electronic structure calculations are performed to determine the stable low-energy conformations of these complexes. Geometry optimizations, frequency analyses, and IR spectra of these species are determined at the B3LYP/6-311+G(d,p) level of theory. Single-point energies are calculated at the B3LYP/6-311+G(2d,2p) level of theory to determine the relative stabilities of these conformations. Comparison of the measure IRMPD action spectra and computed linear IR spectra enable the conformations accessed in the experiments to be elucidated. For both cytosine nucleosides, tridentate binding of the Na+ cation to the O2, O4' and O5' atoms of the nucleobase and sugar is observed. Present results for the sodium cationized nucleosides are compared to results for the analogous protonated forms of these nucleosides to elucidate the effects of multiple chelating interactions with the sodium cation vs. hydrogen bonding interactions in the protonated systems on the structures and stabilities of these nucleosides.

  8. Post-Translational Phosphorylation of Serine 74 of Human Deoxycytidine Kinase Favors the Enzyme Adopting the Open Conformation Making It Competent for Nucleoside Binding and Release

    SciTech Connect

    Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2012-03-26

    Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

  9. Long-term exposure to estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells

    PubMed Central

    Chang, Yu-Wei

    2017-01-01

    Chemotherapy is the most common clinical option for treatment of breast cancer. However, the efficacy of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen responsive and the levels of ovarian estrogen vary among the breast cancer patients primarily between pre- and post-menopausal age. Whether this age-dependent variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is not known. Therefore, the objective of this study was to evaluate the effects of natural estrogen 17 beta-estradiol (E2) on the efficacy of chemotherapeutic drugs in breast cancer cells. Estrogen responsive MCF-7 and T47D breast cancer cells were long-term exposed to 100 pg/ml estrogen, and using these cells the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased sensitivities of doxorubicin and cisplatin in estrogen-exposed MCF-7 and T47D cells as compared to their respective control cells. Gene expression analysis of cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-aza-2-deoxycytidine and Trichostatin A prior to doxorubicin and cisplatin treatments. The 5-aza-2 deoxycytidine pre-treatment significantly decreased the estrogen-induced efficacy of doxorubicin and cisplatin, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In summary, the results of this study revealed that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the chemotherapy strategies for treatment of breast

  10. Relationship Between Chromatin Structure and Sensitivity to Molecularly Targeted Auger Electron Radiation Therapy

    SciTech Connect

    Terry, Samantha Y.A.

    2012-07-15

    Purpose: The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated. Methods and Materials: Chromatin structure was altered in MDA-MB-468 and 231-H2N human breast cancer cells by suberoylanilide hydroxamic acid (SAHA), 5-aza-2-deoxycytidine, or hypertonic treatment. The extent and duration of chromatin structural changes were evaluated using the micrococcal nuclease assay. DNA damage ({gamma}H2AX assay) and clonogenic survival were evaluated after exposure to {sup 111}In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of {sup 111}In-DTPA-hEGF after chromatin modification was investigated in cell fractionation experiments. Results: Chromatin remained condensed for up to 20 minutes after NaCl and in a relaxed state 24 hours after SAHA treatment. The number of {gamma}H2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 {mu}M) compared with IR alone (16 {+-} 0.6 and 14 {+-} 0.3 vs. 12 {+-} 0.4 and 11 {+-} 0.2, respectively). More {gamma}H2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to {sup 111}In-DTPA-hEGF (6 MBq/{mu}g) plus SAHA vs. {sup 111}In-DTPA-hEGF alone (11 {+-} 0.3 and 12 {+-} 0.7 vs. 9 {+-} 0.4 and 7 {+-} 0.3, respectively). 5-aza-2-deoxycytidine enhanced the DNA damage caused by IR and {sup 111}In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 {mu}M) vs. IR alone (0.6% {+-} 0.01 and 0.3% {+-} 0.2 vs. 5.8% {+-} 0.2 and 2% {+-} 0.1, respectively) and after {sup 111}In-DTPA-hEGF plus SAHA compared to {sup 111}In-DTPA-hEGF alone (21% {+-} 0.4% and 19% {+-} 4.6 vs. 33% {+-} 2.3 and 32% {+-} 3.7). SAHA did not affect {sup 111}In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer {gamma}H2AX foci per cell

  11. Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

    SciTech Connect

    Putnik, Milica; Zhao, Chunyan; Gustafsson, Jan-Ake; Dahlman-Wright, Karin

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of

  12. Epidrug-induced upregulation of functional somatostatin type 2 receptors in human pancreatic neuroendocrine tumor cells.

    PubMed

    Veenstra, Marije J; van Koetsveld, Peter M; Dogan, Fadime; Farrell, William E; Feelders, Richard A; Lamberts, Steven W J; de Herder, Wouter W; Vitale, Giovanni; Hofland, Leo J

    2016-05-19

    Somatostatin receptors are a pivotal target for treatment of pancreatic neuroendocrine tumors (pNET), either with somatostatin analogues (SSA) or radiolabeled SSA. The highest affinity target for the most commonly used SSA is the somatostatin receptor type 2 (sst2). An important factor that may complicate treatment efficacy, is the variable number of receptors expressed on pNETs. Gene expression is subject to complex regulation, in which epigenetics has a central role. In this study we explored the possible role of epigenetic modifications in the variations in sst2 expression levels in two human pNET cell lines, BON-1 and QGP-1. We found upregulation of sst2 mRNA after treatment with the epidrugs 5-aza-2'-deoxycytidine (5-aza-dC) and valproic acid (VPA), an increased uptake of radiolabeled octreotide, as well as increased sensitivity to the SSA octreotide in functional cAMP inhibition. At epigenetic level we observed low methylation levels of the sst2 gene promoter region irrespective of expression. Activating histone mark H3K9Ac can be regulated with epidrug treatment, with an angle of effect corresponding to the effect on mRNA expression. Repressive histone mark H3K27me3 is not regulated by either 5-aza-dC or VPA. We conclude that epidrug treatment, in particular with combined 5-aza-dC and VPA treatment, might hold promise for improving and adding to current SSA treatment strategies of patients with pNETs.

  13. Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Inoue, Kazuyuki; Hayashi, Hideki; Tsuji, Daiki; Mizugaki, Michinao; Itoh, Kunihiko

    2010-11-01

    DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

  14. Detection of endogenous DNA adducts, O-carboxymethyl-2'-deoxyguanosine and 3-ethanesulfonic acid-2'-deoxycytidine, in the rat stomach after duodenal reflux.

    PubMed

    Terasaki, Masaru; Totsuka, Yukari; Nishimura, Koichi; Mukaisho, Ken-Ichi; Chen, Kuan-Hao; Hattori, Takanori; Takamura-Enya, Takeji; Sugimura, Takashi; Wakabayashi, Keiji

    2008-09-01

    The endogenous DNA adducts O(6)-carboxymethyl-deoxyguanosine (O(6)-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC) are produced from N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NO-GCA) and N-nitrosotaurocholic acid (NO-TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P-postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O(6)-CM-dG found at 4 and 8 weeks after surgery were 40.9 +/- 9.4 and 56.3 +/- 3.2 per 10(8) nucleotides, respectively, whereas the sham operation groups had values of 5.8 +/- 2.3 and 5.9 +/- 0.5 per 10(8) nucleotides. Moreover, adduct spots corresponding to 3-ESA-dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four-fold elevated at 11.2 +/- 1.0 and 8.9 +/- 1.0 per 10(8) nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine- 4-carboxylic acid (thioproline, TPRO), the levels of O(6)-CM-dG and 3-ESA-dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO-TCA and NO-GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery.

  15. A Mathematical Model for Scanning and Catalysis on Single-stranded DNA, Illustrated with Activation-induced Deoxycytidine Deaminase*♦

    PubMed Central

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2013-01-01

    We formulated a master equation-based mathematical model to analyze random scanning and catalysis for enzymes that act on single-stranded DNA (ssDNA) substrates. Catalytic efficiencies and intrinsic scanning distances are deduced from the distribution of positions and gap lengths between a series of catalytic events occurring over time, which are detected as point mutations in a lacZα-based reporter sequence containing enzyme target motifs. Mathematical analysis of the model shows how scanning motions become separable from the catalysis when the proper statistical properties of the mutation pattern are used to interpret the readouts. Two-point correlations between all catalytic events determine intrinsic scanning distances, whereas gap statistics between mutations determine their catalytic efficiencies. Applying this model to activation-induced deoxycytidine deaminase (AID), which catalyzes C→U deaminations processively on ssDNA, we have established that deaminations of AGC hot motifs occur at a low rate, ∼0.03 s−1, and low efficiency, ∼3%. AID performs random bidirectional movements for an average distance of 6.2 motifs, at a rate of about 15 nucleotides per second, and “dwells” at a motif site for 2.7 s while bound >4 min to the same DNA molecule. These results provide new and important insights on how AID may be optimized for generating mutational diversity in Ig genes, and we discuss how the properties of AID acting freely on a “naked” ssDNA relate to the constrained action of AID during transcription-dependent somatic hypermutation and class-switch recombination. PMID:23979486

  16. Protonation of deoxycytidine residues in dC4 tetraloops: UV spectrophotometric study of dC10 and d(A14C4T14).

    PubMed

    Raukas, E; Kooli, K

    2003-06-01

    It is shown that component analysis could be applied to study the UV difference spectra of cytidine oligomers and hairpin oligonucleotides with cytidines in the loop region in order to account for the melting and titration results in terms of cytidine stacking and protonation. Upon acid titration, the dC(10) oligomer undergoes cooperative conformational transition at pH 6.3 accompanied by protonation and formation of the i-structure with half of the residues protonated. The stability of the hemiprotonated structure increases with decreasing pH, the i-structure persisting still in the region of pHdeoxycytidine tetraloop is attained at pH 5.0. Simultaneously, the stacking interactions of cytidine residues reach the maximum at this pH with two residues stacked, and thereafter decline again. Only marginal stabilization of the oligomer hairpin (DeltaT(m)=1.5 degrees C) is found to accompany the formation of this single hemiprotonated dC.dC(+) base pair. We propose that at pH 5 the cytidines of the dC(4) loop form a hemiprotonated dC.dC(+) pair stacked with the last dA.dT base pair of the hairpin stem.

  17. Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine.

    PubMed

    Li, Deyu; Fedeles, Bogdan I; Singh, Vipender; Peng, Chunte Sam; Silvestre, Katherine J; Simi, Allison K; Simpson, Jeffrey H; Tokmakoff, Andrei; Essigmann, John M

    2014-08-12

    Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

  18. Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe.

    PubMed

    Zhang, Hongge; Wang, Minjuan; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2011-05-15

    A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F(0)=2.73 C (μM)+1.14 (R=0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N=3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.

  19. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: adenosylcobalamin destruction and formation of a nucleotide-based radical.

    PubMed

    Lohman, Gregory J S; Gerfen, Gary J; Stubbe, Joanne

    2010-02-23

    Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in <2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C(419) (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u ]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F(2)CTP and the second with AdoCbl destruction. To determine the fate of [1'-(3)H]F(2)CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH(4) (NaB(2)H(4)) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

  20. All-trans retinoic acid enhances gemcitabine cytotoxicity in human pancreatic cancer cell line AsPC-1 by up-regulating protein expression of deoxycytidine kinase.

    PubMed

    Kuroda, Hiroki; Tachikawa, Masanori; Uchida, Yasuo; Inoue, Koetsu; Ohtsuka, Hideo; Ohtsuki, Sumio; Unno, Michiaki; Terasaki, Tetsuya

    2017-02-12

    We previously showed that gemcitabine resistance in pancreatic cancer chemotherapy correlates with suppressed expression of deoxycytidine kinase (dCK), which catalyzes the rate-limiting step of gemcitabine activation. The purpose of the present study was to find a drug that might be useful to enhance the cytotoxicity of gemcitabine by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cell line AsPC-1. Screening of 40 prescription drugs identified 35 with no intrinsic cytotoxicity towards AsPC-1 cells. When AsPC-1 cells were pre-incubated with these drugs and then incubated with gemcitabine, we found that all-trans retinoic acid (ATRA) significantly decreased the viability by 28% compared with that of non-treated cells. Luciferase assay showed that ATRA transactivated the DCK promoter in AsPC-1 cells by about 2-fold compared with the untreated control, and an increase of dCK protein expression was confirmed by immunoblotting. ATRA decreased the half-maximal inhibitory concentration (IC50) of gemcitabine by 2.8-fold (ATRA-non-treated cells, 28.8nM; ATRA-treated cells, 10.0nM). The ATRA concentration of 0.03μM was sufficient to enhance gemcitabine cytotoxicity, and the effect was well maintained in the concentration range from 0.03 to 50μM. These results indicate that ATRA enhances gemcitabine cytotoxicity by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cells.

  1. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine

    PubMed Central

    Ligasová, Anna; Liboska, Radek; Friedecký, David; Mičová, Kateřina; Adam, Tomáš; Oždian, Tomáš; Rosenberg, Ivan; Koberna, Karel

    2016-01-01

    5-Ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA. PMID:26740587

  2. Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

    PubMed Central

    Magewu, A N; Jones, P A

    1994-01-01

    Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine. Images PMID:8196660

  3. Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

    PubMed

    Pérez-Campo, Flor M; May, Tobias; Zauers, Jeannette; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Berciano, María T; Lafarga, Miguel; Riancho, José A

    2017-03-01

    Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

  4. Inhibition of DNA Methylation Suppresses Intestinal Tumor Organoids by Inducing an Anti-Viral Response.

    PubMed

    Saito, Yoshimasa; Nakaoka, Toshiaki; Sakai, Kasumi; Muramatsu, Toshihide; Toshimitsu, Kohta; Kimura, Masaki; Kanai, Takanori; Sato, Toshiro; Saito, Hidetsugu

    2016-05-04

    Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of Apc(Min/+) (Min) mice and subjected them to 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.

  5. Time-course gene profiling and networks in demethylated retinoblastoma cell line.

    PubMed

    Malusa, Federico; Taranta, Monia; Zaki, Nazar; Cinti, Caterina; Capobianco, Enrico

    2015-09-15

    Retinoblastoma, a very aggressive cancer of the developing retina, initiatiates by the biallelic loss of RB1 gene, and progresses very quickly following RB1 inactivation. While its genome is stable, multiple pathways are deregulated, also epigenetically. After reviewing the main findings in relation with recently validated markers, we propose an integrative bioinformatics approach to include in the previous group new markers obtained from the analysis of a single cell line subject to epigenetic treatment. In particular, differentially expressed genes are identified from time course microarray experiments on the WERI-RB1 cell line treated with 5-Aza-2'-deoxycytidine (decitabine; DAC). By inducing demethylation of CpG island in promoter genes that are involved in biological processes, for instance apoptosis, we performed the following main integrative analysis steps: i) Gene expression profiling at 48h, 72h and 96h after DAC treatment; ii) Time differential gene co-expression networks and iii) Context-driven marker association (transcriptional factor regulated protein networks, master regulatory paths). The observed DAC-driven temporal profiles and regulatory connectivity patterns are obtained by the application of computational tools, with support from curated literature. It is worth emphasizing the capacity of networks to reconcile multi-type evidences, thus generating testable hypotheses made available by systems scale predictive inference power. Despite our small experimental setting, we propose through such integrations valuable impacts of epigenetic treatment in terms of gene expression measurements, and then validate evidenced apoptotic effects.

  6. Time-course gene profiling and networks in demethylated retinoblastoma cell line

    PubMed Central

    Malusa, Federico; Taranta, Monia; Zaki, Nazar; Cinti, Caterina; Capobianco, Enrico

    2015-01-01

    Retinoblastoma, a very aggressive cancer of the developing retina, initiatiates by the biallelic loss of RB1 gene, and progresses very quickly following RB1 inactivation. While its genome is stable, multiple pathways are deregulated, also epigenetically. After reviewing the main findings in relation with recently validated markers, we propose an integrative bioinformatics approach to include in the previous group new markers obtained from the analysis of a single cell line subject to epigenetic treatment. In particular, differentially expressed genes are identified from time course microarray experiments on the WERI-RB1 cell line treated with 5-Aza-2′-deoxycytidine (decitabine; DAC). By inducing demethylation of CpG island in promoter genes that are involved in biological processes, for instance apoptosis, we performed the following main integrative analysis steps: i) Gene expression profiling at 48h, 72h and 96h after DAC treatment; ii) Time differential gene co-expression networks and iii) Context-driven marker association (transcriptional factor regulated protein networks, master regulatory paths). The observed DAC-driven temporal profiles and regulatory connectivity patterns are obtained by the application of computational tools, with support from curated literature. It is worth emphasizing the capacity of networks to reconcile multi-type evidences, thus generating testable hypotheses made available by systems scale predictive inference power. Despite our small experimental setting, we propose through such integrations valuable impacts of epigenetic treatment in terms of gene expression measurements, and then validate evidenced apoptotic effects. PMID:26143641

  7. Intracellular cytarabine triphosphate production correlates to deoxycytidine kinase/cytosolic 5'-nucleotidase II expression ratio in primary acute myeloid leukemia cells.

    PubMed

    Yamauchi, Takahiro; Negoro, Eiju; Kishi, Shinji; Takagi, Kazutaka; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

    2009-06-15

    Cytarabine (ara-C) is the key agent for treating acute myeloid leukemia (AML). After being transported into leukemic cells by human equilibrative nucleoside transporter 1 (hENT1), ara-C is phosphorylated to ara-C triphosphate (ara-CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase II (cN-II) are associated with the production of ara-CTP. Because ara-C's cytotoxicity depends on ara-CTP production, parameters that are most related to ara-CTP formation would predict ara-C sensitivity and the clinical outcome of ara-C therapy. The present study focused on finding any correlation between the capacity to produce ara-CTP and ara-C-metabolizing factors. In vitro ara-CTP production, mRNA levels of hENT1, dCK, and cN-II, and ara-C sensitivity were evaluated in 34 blast samples from 33 leukemic patients including 26 with AML. A large degree of heterogeneity was seen in the capacity to produce ara-CTP and in mRNA levels of hENT1, dCK, and cN-II. Despite the lack of any association between each of the transcript levels and ara-CTP production, the ratio of dCK/cN-II transcript levels correlated significantly with the amount of ara-CTP among AML samples. The HL-60 cultured leukemia cell line and its three ara-C-resistant variants (HL-60/R1, HL-60/R2, HL-60/R3), which were 8-, 10-, and 500-fold more resistant than HL-60, respectively, were evaluated similarly. The dCK/cN-II ratio was again proportional to ara-CTP production and to ara-C sensitivity. The dCK/cN-II ratio may thus predict the capacity for ara-CTP production and ultimately, ara-C sensitivity in AML.

  8. Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2

    PubMed Central

    Yaghi, Layale; Poras, Isabelle; Simoes, Renata T.; Donadi, Eduardo A.; Tost, Jörg; Daunay, Antoine; de Almeida, Bibiana Sgorla; Carosella, Edgardo D.; Moreau, Philippe

    2016-01-01

    HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at −966 bp in the 5′UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus. PMID:27577073

  9. Antenatal Hypoxia Induces Epigenetic Repression of Glucocorticoid Receptor and Promotes Ischemic-Sensitive Phenotype in the Developing Heart

    PubMed Central

    Xiong, Fuxia; Lin, Thant; Song, Minwoo; Ma, Qingyi; Martinez, Shannalee R.; Lv, Juanxiu; MataGreenwood, Eugenia; Xiao, Daliao; Xu, Zhice; Zhang, Lubo

    2016-01-01

    Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at −4408 and −3896 and Sp1 binding sites at −3425 and −3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2’-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart. PMID:26779948

  10. Methylation status of oestrogen receptor alpha-A: a predictor of prognosis in leukaemias.

    PubMed

    Yao, Jie; Zhang, Xiao-Bing; Zhang, Xiao-li; Fu, Wei-Ling

    2010-03-12

    Many studies have shown that epigenetic regulation of ERs (oestrogen receptors) plays a key role in the pathogenesis of leukaemia. In the present study, it was found that the methylated status of ERalpha-A might serve as an epigenetic biomarker of leukaemias. In this study, the protein expression and cell apoptosis, cycle, proliferation and viability with and without 5-aza-dC (5-aza-2'-deoxycytidine) were evaluated with Western blotting, 3H-TdR (3H-thymidine) incorporation, propidium iodide staining and Trypan Blue staining respectively. The protein expression of ERalpha was significantly enhanced in all leukaemic cell lines using treatment with the DNA demethylation reagent 5-aza-dC. However, no obvious change in the protein expression of ERbeta takes place with 5-aza-dC. And with 5-aza-dC, cell apoptosis, cell cycle, cell proliferation and viability were all inhibited significantly. We also tracked 40 cases of leukaemias with ERalpha-A methylation (95%; 38 of 40) to observe the prognosis 1 year after chemotherapy treatment. The patients with ERalpha-A methylation have no obvious symptomatic relief; however, two patients without ERalpha-A methylation have obtained effective relief. This result suggested that ERalpha plays a significant role in leukaemogenesis, and the methylated status of ERalpha-A not only might serve as an epigenetic biomarker of leukaemias for diagnosis, but also has the potential to serve as a predictor of prognosis in leukaemias.

  11. DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium.

    PubMed

    Plachot, Cedric; Lelièvre, Sophie A

    2004-08-01

    Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.

  12. The early epigenetic response to ozone: impacts on DNA ...

    EPA Pesticide Factsheets

    Epigenetics have been increasingly recognized as a mechanism linking environment and gene expression. Despite awareness of the role of DNA methylation and hydroxymethylation as potential drivers of the response to air pollutants, very little work has been performed investigating the direct epigenetic effects following exposure to ambient air pollution. Thus the purpose of this study was to investigate the early epigenetic response to ozone in comparison to the epigenetic modifier 5-aza-2'-deoxycytidine (5-Aza) in rats. 12 week old, male Long-Evans rats (n=16) were exposed to 4 hours of whole-body 1.0 ppm ozone or air and immediately euthanized. A subset of animals were additionally treated with 5-Aza (n=16) to serve as an epigenetic control to ozone exposure. Neither 5-Aza nor ozone by itself induced changes to the global methylome or hydroxmethylome of the lung measured by ELISA. Despite this finding, ozone exposure induced a significant increase in the activity of the DNA methyltransferase enzymes in the lung which was reversed with 5-Aza treatment. Interestingly, a significant interaction between 5-Aza treatment and ozone exposure was found in a large array of data. The interaction between 5-Aza and ozone produced indicators of pulmonary edema and elevated lung damage. Along with these adverse changes, expression of major epigenetic enzymes (Tet 1-3, Dnmt3 a-b) were found to be perturbed in both the lung and hepatic tissues. While ozone exposure appears to in

  13. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

    PubMed Central

    Yang, Allen S.; Estécio, Marcos R. H.; Doshi, Ketan; Kondo, Yutaka; Tajara, Eloiza H.; Issa, Jean-Pierre J.

    2004-01-01

    We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation. PMID:14973332

  14. Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2.

    PubMed

    Yaghi, Layale; Poras, Isabelle; Simoes, Renata T; Donadi, Eduardo A; Tost, Jörg; Daunay, Antoine; de Almeida, Bibiana Sgorla; Carosella, Edgardo D; Moreau, Philippe

    2016-09-27

    HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2'deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at -966 bp in the 5'UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

  15. Effect of DNA methylation profile on OATP3A1 and OATP4A1 transcript levels in colorectal cancer.

    PubMed

    Rawłuszko-Wieczorek, Agnieszka Anna; Horst, Nikodem; Horbacka, Karolina; Bandura, Artur Szymon; Świderska, Monika; Krokowicz, Piotr; Jagodziński, Paweł Piotr

    2015-08-01

    Epidemiological studies indicate that 17β-estradiol (E2) prevents colorectal cancer (CRC). Organic anion transporting polypeptides (OATPs) are involved in the cellular uptake of various endogenous and exogenous substrates, including hormone conjugates. Because transfer of estrone sulfate (E1-S) can contribute to intra-tissue conversion of estrone to the biologically active form -E2, it is evident that the expression patterns of OATPs may be relevant to the analysis of CRC incidence and therapy. We therefore evaluated DNA methylation and transcript levels of two members of the OATP family, OATP3A1 and OATP4A1, that may be involved in E1-S transport in colorectal cancer patients. We detected a significant reduction in OATP3A1 and a significant increase in OATP4A1 mRNA levels in cancerous tissue, compared with histopathologically unchanged tissue (n=103). Moreover, we observed DNA hypermethylation in the OATP3A1 promoter region in a small subset of CRC patients and in HCT116 and Caco-2 colorectal cancer cell lines. We also observed increased OATP3A1 transcript following treatment with 5-aza-2-deoxycytidine and sodium butyrate. The OATP4A1 promoter region was hypomethylated in analyzed tissues and CRC cell lines and was not affected by these treatments. Our results suggest a potential mechanism for OATP3A1 downregulation that involves DNA methylation during colorectal carcinogenesis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  16. Sustained IL-6/STAT-3 Signaling in Cholangiocarcinoma Cells due to SOCS-3 Epigenetic Silencing

    PubMed Central

    Isomoto, Hajime; Mott, Justin L.; Kobayashi, Shogo; Werneburg, Nathan W.; Bronk, Steve F.; Haan, Serge; Gores, Gregory J.

    2008-01-01

    Background and aims IL-6 mediated STAT-3 phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced Mcl-1 expression and resistance to apoptosis. Because SOCS-3 controls the IL-6/STAT-3 signaling pathway by a classic feedback loop, the aims of this study were to examine SOCS-3 regulation in human cholangiocarcinoma. Methods SOCS-3 expression was assessed in human cholangiocarcinoma tissue and the Mz-ChA-1 and CCLP1 human cholangiocarcinoma cell lines. Results An inverse correlation was observed between phospho-STAT-3 and SOCS-3 protein expression in cholangiocarcinoma. In those cancers failing to express SOCS-3, extensive methylation of the SOCS-3 promoter was demonstrated in tumor but not in paired non-tumor tissue. Likewise, methylation of the socs-3 promoter was also identified in two cholangiocarcinoma cell lines. Treatment with a demethylating agent, 5-aza-2′-deoxycytidine (DAC), restored IL-6 induction of SOCS-3, terminated the phospho-STAT-3 response, and reduced cellular levels of Mcl-1. Enforced expression of SOCS-3 also reduced IL-6 induction of phospho-STAT-3 and Mcl-1. Either DAC treatment or enforced SOCS-3 expression sensitized the cells to TRAIL-mediated apoptosis. Conclusion SOCS-3 epigenetic silencing is responsible for sustained IL-6/STAT-3 signaling and enhanced Mcl-1 expression in cholangiocarcinoma. PMID:17241887

  17. Gene structure and transcription in mouse cells with extensively demethylated DNA.

    PubMed Central

    Michalowsky, L A; Jones, P A

    1989-01-01

    In previous work, three clonal cell lines with extremely low DNA methylation levels were derived by multiple consecutive treatments of C3H 10T1/2 C18 (10T1/2) cells with 5-aza-2'-deoxycytidine (5-aza-CdR). In this study we examined the methylation status of genes in these three methyl-deficient clones to assess the specificity of the induced hypomethylation. Complete demethylation of virtually all 5'-CCGG-3' sites was observed in four genes examined, but some sites common to all three clones were persistently methylated even after further exhaustive 5-aza-CdR treatment. Thus, there is a subset of methylation sites within these cells which can never be stably demethylated. The extensive demethylation was not always associated with changes in the level of RNA expression of the genes examined but was strongly correlated with an altered chromatin structure of the unexpressed alpha 1-globin gene and the muscle determination gene MyoD1. These results provide a direct correlation between hypomethylation and the induction of a transcriptionally competent chromatin state. Images PMID:2471061

  18. Antenatal hypoxia induces epigenetic repression of glucocorticoid receptor and promotes ischemic-sensitive phenotype in the developing heart.

    PubMed

    Xiong, Fuxia; Lin, Thant; Song, Minwoo; Ma, Qingyi; Martinez, Shannalee R; Lv, Juanxiu; MataGreenwood, Eugenia; Xiao, Daliao; Xu, Zhice; Zhang, Lubo

    2016-02-01

    Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at -4408 and -3896 and Sp1 binding sites at -3425 and -3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2'-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart.

  19. Inhibition of EHMT2/G9a epigenetically increases the transcription of Beclin-1 via an increase in ROS and activation of NF-κB

    PubMed Central

    Park, Sang Eun; Yi, Hye Jin; Suh, Nayoung; Park, Yun-Yong; Koh, Jae-Young; Jeong, Seong-Yun; Cho, Dong-Hyung; Kim, Choung-Soo; Hwang, Jung Jin

    2016-01-01

    We previously reported that BIX-01294 (BIX), a small molecular inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a), induces reactive oxygen species (ROS)-dependent autophagy in MCF-7 cells. Herein, we analyzed the epigenetic mechanism that regulates the transcription of Beclin-1, a tumor suppressor and an autophagy-related gene (ATG). Inhibition of EHMT2 reduced dimethylation of lysine 9 on histone H3 (H3K9me2) and dissociated EHMT2 and H3K9me2 from the promoter of Beclin-1. To this promoter, RNA polymerase II and nuclear factor kappa B (NF-κB) were recruited in a ROS-dependent manner, resulting in transcriptional activation. Moreover, treatment with BIX reversed the suppression of Beclin-1 by the cooperative action of EHMT2 and DNA methyltransferase 1 (DNMT1). Accordingly, a combination treatment with BIX and 5-Aza-2′-deoxycytidine (5-Aza-Cd), a DNMT1 inhibitor, exerted a synergistic effect on Beclin-1 expression. Importantly, high levels of EHMT2 expression showed a significant association with low levels of Beclin-1 expression, which was related to a poor prognosis. These findings suggest that EHMT2 can directly repress Beclin-1 and that the inhibition of EHMT2 may be a useful therapeutic approach for cancer prevention by activating autophagy. PMID:27174920

  20. KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients

    PubMed Central

    Chen, Shao-Qin; Chen, Zhi-Hua; Lin, Su-Yong; Dai, Qi-Bao; Fu, Leng-Xi; Chen, Rui-Qing

    2014-01-01

    AIM: To examine the effect of aberrant methylation of the KISS1 promoter on the development of colorectal cancer (CRC) and to investigate reversing aberrant methylation of the KISS1 promoter as a potential therapeutic target. METHODS: KISS1 promoter methylation, mRNA expression and protein expression were detected by methylation-specific polymerase chain reaction (PCR), real-time quantitative PCR and Western blotting, respectively, in 126 CRC tissues and 142 normal colorectal tissues. Human CRC cells with KISS1 promoter hypermethylation and poor KISS1 expression were treated in vitro with 5-aza-2’-deoxycytidine (5-Aza-CdR). After treatment, KISS1 promoter methylation, KISS1 mRNA and protein expression and cell migration and invasion were evaluated. RESULTS: Hypermethylation of KISS1 occurred frequently in CRC samples (83.1%, 105/126), but was infrequent in normal colorectal tissues (6.34%, 9/142). Moreover, KISS1 methylation was associated with tumor differentiation, the depth of invasion, lymph node metastasis and distant metastasis (P < 0.001). KISS1 methylation was also associated with low KISS1 expression (P < 0.001). Furthermore, we observed re-expression of the KISS1 gene and decreased cell migration after 5-Aza-CdR treatment in a CRC cell line. CONCLUSION: These data suggest that KISS1 is down-regulated in cancer tissues via promoter hypermethylation and therefore may represent a candidate target for treating metastatic CRC. PMID:25110434

  1. Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer

    PubMed Central

    Liu, Bangqing; Song, Jianfei; Luan, Jiaqiang; Sun, Xiaolin; Bai, Jian; Wang, Haiyong; Li, Angui; Zhang, Lifei; Feng, Xiaoyan

    2016-01-01

    DNA methylation is an epigenetic DNA modification catalyzed by DNA methyltransferase 1 (DNMT1). The purpose of this study was to investigate DNMT1 gene and protein expression and the effects of methylation status on tumor suppressor genes in human non-small cell lung cancer (NSCLC) cell lines grown in vitro and in vivo. Human lung adenocarcinoma cell lines, A549 and H838, were grown in vitro and inoculated subcutaneously into nude mice to form tumors and were then treated with the DNA methylation inhibitor, 5-aza-2′-deoxycytidine, with and without treatment with the benzamide histone deacetylase inhibitor, entinostat (MS-275). DNMT1 protein expression was quantified by Western blot. Promoter methylation status of tumor suppressor genes (RASSF1A, ASC, APC, MGMT, CDH13, DAPK, ECAD, P16, and GATA4) was evaluated by methylation-specific polymerase chain reaction. Methylation status of the tumor suppressor genes was regulated by the DNMT1 gene, with the decrease of DNMT1 expression following DNA methylation treatment. Demethylation of tumor suppressor genes (APC, ASC, and RASSF1A) restored tumor growth in nude mice. The results of this study support a role for methylation of DNA as a potential epigenetic clinical biomarker of prognosis or response to therapy and for DNMT1 as a potential therapeutic target in NSCLC. PMID:27190263

  2. Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

    PubMed Central

    Ostrow, Kimberly Laskie; Park, Hannah Lui; Hoque, Mohammad Obaidul; Kim, Myoung Sook; Liu, Junwei; Argani, Pedram; Westra, William; Van Criekinge, Wim; Sidransky, David

    2011-01-01

    Purpose Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2′-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer. Experimental Design Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes. Results Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P < 0.0001, Fisher’s exact test). Conclusions This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease. PMID:19228724

  3. An endoparasitoid wasp influences host DNA methylation

    PubMed Central

    Kumar, Sunil; Kim, Yonggyun

    2017-01-01

    Parasitism by endoparasitoid wasps changes the expression of various host genes, and alters host immune and developmental processes. However, it is not clearly understood how parasitism changes host gene expression in a whole genome scale. This study focused on an epigenetic control of Cotesia plutellae, an endoparasitoid wasp, against its host, Plutella xylostella. Two DNA methyltransferases (DNMT-1 and DNMT-2) are encoded in the genome of P. xylostella. In addition, methyl-binding domain proteins (MBDs) and DNA demethylation factor, ten-eleven translation protein (TET) are encoded. DNA methylation of P. xylostella genomic DNA was confirmed by restriction digestion with Gla I specific to 5-methylcytosine. DNA methylation intensity in parasitized (P) larvae was decreased compared to that in nonparasitized (NP) larvae, especially at late parasitic stage, at which expression levels of both DNMT-1 and DNMT-2 were also decreased. DNA demethylation of P. xylostella was confirmed in both NP and P larvae by restriction digestion with PvuRts1I recognizing 5-hydroxymethyl cytosine. Parasitism also suppressed expression levels of TET and MBDs. Treatment of 5-aza-2′-deoxycytidine (AZA) reduced DNA methylation intensity of NP larvae, causing suppression of hemocyte-spreading behavior and delay of immature development. RNA interference of DNMT-1 or DNMT-2 mimicked the adverse effects of AZA. PMID:28230192

  4. PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells

    SciTech Connect

    Gonen, Nitzan; Bram, Eran E.; Assaraf, Yehuda G.

    2008-11-28

    The proton-coupled folate transporter (PCFT/SLC46A1) displays optimal and prominent folate and antifolate transport activity at acidic pH in human carcinoma cells but poor activity in leukemia cells. Consistently herein, human leukemia cell lines expressed poor PCFT transcript levels, whereas various carcinoma cell lines showed substantial PCFT gene expression. We identified a CpG island with high density at nucleotides -200 through +100 and explored its role in PCFT promoter silencing. Leukemia cells with barely detectable PCFT transcripts consistently harbored 85-100% methylation of this CpG island, whereas no methylation was found in carcinoma cells. Treatment with 5-Aza-2'-deoxycytidine which induced demethylation but not with the histone deacetylase inhibitor trichostatin A, restored 50-fold PCFT expression only in leukemia cells. These findings constitute the first demonstration of the dominant epigenetic silencing of the PCFT gene in leukemia cells. The potential translational implications of the restoration of PCFT expression in chemotherapy of leukemia are discussed.

  5. Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells

    PubMed Central

    Kim, Ji Tae; Li, Jing; Song, Jun; Lee, Eun Y.; Weiss, Heidi L.; Townsend, Courtney M.; Evers, B. Mark

    2015-01-01

    Neurotensin (NTS), localized predominantly to the small bowel, stimulates the growth of a variety of cancers, including neuroendocrine tumors (NETs), mainly through its interaction with the high-affinity NTS receptor 1 (NTSR1). Here, we observed increased expression of NTSR1 in almost all tested clinical NET samples, but not in normal tissues. Through RT-PCR analysis, we found that the expression of NTSR1 and NTSR2 was either variable (NTSR1) or absent (NTSR2) in human NET cell lines. In contrast, NTSR3 and NTS were expressed in all NET cells. Treatment with 5-aza-2′-deoxycytidine, a demethylating agent, increased levels of NTSR1 and NTSR2 suggesting that DNA methylation contributes to NTSR1/2 expression patterns, which was confirmed by methylation analyses. In addition, we found that knockdown of NTSR1 decreased proliferation, expression levels of growth-related proteins, and anchorage-independent growth of BON human carcinoid cells. Moreover, stable silencing of NTSR1 suppressed BON cell growth, adhesion, migration and invasion. Our results show that high expression of NTSR1 is found in clinical NETs and that promoter methylation is an important mechanism controlling the differential expression of NTSR1 and silencing of NTSR2 in NET cells. Furthermore, knockdown of NTSR1 in BON cells suppressed oncogenic functions suggesting that NTSR1 contributes to NET tumorigenesis. PMID:26298774

  6. Inactivation of the candidate tumor suppressor par-4 in endometrial cancer.

    PubMed

    Moreno-Bueno, Gema; Fernandez-Marcos, Pablo J; Collado, Manuel; Tendero, Mercedes J; Rodriguez-Pinilla, Socorro M; Garcia-Cao, Isabel; Hardisson, David; Diaz-Meco, Maria T; Moscat, Jorge; Serrano, Manuel; Palacios, Jose

    2007-03-01

    Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in approximately 40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or beta-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5'-aza-2'-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis.

  7. High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer

    PubMed Central

    Ferguson, Anne T.; Evron, Ella; Umbricht, Christopher B.; Pandita, Tej K.; Chan, Timothy A.; Hermeking, Heiko; Marks, Jeffrey R.; Lambers, Anouk R.; Futreal, P. Andrew; Stampfer, Martha R.; Sukumar, Saraswati

    2000-01-01

    Expression of 14-3-3 σ (σ) is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified σ as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, σ mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at σ such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the σ gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of σ is functionally important, because treatment of σ-non-expressing breast cancer cell lines with the drug 5-aza-2′-deoxycytidine resulted in demethylation of the gene and synthesis of σ mRNA. Breast cancer cells lacking σ expression showed increased number of chromosomal breaks and gaps when exposed to γ-irradiation. Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of σ expression are the most consistent molecular alterations in breast cancer identified so far. PMID:10811911

  8. In vitro study of human mutL homolog 1 hypermethylation in inducing drug resistance of esophageal carcinoma.

    PubMed

    Cao, Y; Chen, Y; Huang, Y; Liu, Z; Li, G

    2017-05-01

    Aberrant promoter methylation of tumor suppressor gene can inhibit corresponding protein expression and promote carcinogenesis. Many studies have demonstrated that human mutL homolog 1(hMLH1) promoter methylation is correlated with occurrence and progression of multiple types of tumors. However, its correlation with esophageal carcinoma drug resistance is still unknown. To confirm methylation status of hMLH1 promoter in drug-resistance cell line of esophageal carcinoma, further confirm whether hMLH1 promoter methylation is responsible for drug resistance. Two stable esophageal carcinoma drug-resistance cell lines were successfully established by Cisplatin (DDP) concentration increment method; methylation status of hMLH1 promoter, mRNA and protein expression of hMLH1 were detected by methylation-specific PCR (MSP), RT-PCR and western blot, respectively; Drug-resistance ability assay was used to detect drug-resistance ability. Stronger methylation status of hMLH1 promoter, lower hMLH1 mRNA and protein expression were found in both drug-resistance cell lines; after removing methylated bands using 5-aza-2'-deoxycytidine(5-Aza-CdR) in drug-resistance cell lines, hMLH1 mRNA and protein expression were restored and drug-resistance abilities declined nearly by half. hMLH1 promoter hypermethylation plays important roles in esophageal carcinoma drug-resistance and show us the prospect that combination of demethylation treatment with conventional chemotherapy drugs may bring better therapy effect.

  9. Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes

    PubMed Central

    Atilano, Shari R.; Malik, Deepika; Chwa, Marilyn; Cáceres-Del-Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin; Kenney, M. Cristina

    2015-01-01

    Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2′-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. PMID:25964427

  10. Promoter CpG methylation in cancer cells contributes to the regulation of MUC4

    PubMed Central

    Yamada, N; Nishida, Y; Tsutsumida, H; Goto, M; Higashi, M; Nomoto, M; Yonezawa, S

    2009-01-01

    Mucin 4 (MUC4) is a high molecular weight transmembrane mucin that is overexpressed in many carcinomas and is a risk factor associated with a poor prognosis. In this study, we show that the DNA methylation pattern is intimately correlated with MUC4 expression in breast, lung, pancreas and colon cancer cell lines. We mapped the DNA methylation status of 94 CpG sites from −3622 to +29 using MassARRAY analysis that utilises base-specific cleavage of nucleic acids. MUC4-negative cancer cell lines and those with low MUC4 expression (eg, A427) were highly methylated near the transcriptional start site, whereas MUC4-positive cell lines (eg, NCI-H292) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC4-negative cells or those with low MUC4 expression caused elevation of MUC4 mRNA. Our results suggest that DNA methylation in the 5′ flanking region play an important role in MUC4 gene expression in carcinomas of various organs. An understanding of epigenetic changes in MUC4 may contribute to the diagnosis of carcinogenic risk and prediction of outcome in patients with cancer. PMID:19127263

  11. Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.

    PubMed

    Nitzki, Frauke; Tolosa, Ezequiel J; Cuvelier, Nicole; Frommhold, Anke; Salinas-Riester, Gabriela; Johnsen, Steven A; Fernandez-Zapico, Martin E; Hahn, Heidi

    2015-04-20

    Mice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch.

  12. Identification of cis- and trans-acting elements regulating calretinin expression in mesothelioma cells

    PubMed Central

    Kresoja-Rakic, Jelena; Kapaklikaya, Esra; Ziltener, Gabriela; Dalcher, Damian; Santoro, Raffaella; Christensen, Brock C.; Johnson, Kevin C.; Schwaller, Beat; Weder, Walter; Stahel, Rolf A.; Felley-Bosco, Emanuela

    2016-01-01

    Calretinin (CALB2) is a diagnostic marker for epithelioid mesothelioma. It is also a prognostic marker since patients with tumors expressing high calretinin levels have better overall survival. Silencing of calretinin decreases viability of epithelioid mesothelioma cells. Our aim was to elucidate mechanisms regulating calretinin expression in mesothelioma. Analysis of calretinin transcript and protein suggested a control at the mRNA level. Treatment with 5-aza-2′-deoxycytidine and analysis of TCGA data indicated that promoter methylation is not likely to be involved. Therefore, we investigated CALB2 promoter by analyzing ~1kb of genomic sequence surrounding the transcription start site (TSS) + 1 using promoter reporter assay. Deletion analysis of CALB2 proximal promoter showed that sequence spanning the −161/+80bp region sustained transcriptional activity. Site-directed analysis identified important cis-regulatory elements within this −161/+80bp CALB2 promoter. EMSA and ChIP assays confirmed binding of NRF-1 and E2F2 to the CALB2 promoter and siRNA knockdown of NRF-1 led to decreased expression of calretinin. Cell synchronization experiment showed that calretinin expression was cell cycle regulated with a peak of expression at G1/S phase. This study provides the first insight in the regulation of CALB2 expression in mesothelioma cells. PMID:26848772

  13. Down-regulation of BRMS1 by DNA hypermethylation and its association with metastatic progression in triple-negative breast cancer.

    PubMed

    Kong, Bin; Lv, Zhi-Dong; Wang, Yu; Jin, Li-Ying; Ding, Lei; Yang, Zhao-Chuan

    2015-01-01

    Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene in several solid tumors. However, the expression and function of BRMS1 in triple-negative breast cancer (TNBC) have not been reported. In this study, we found that BRMS1 was down-regulation in breast cancer cell lines and primary TNBC, while decreased expression of BRMS1 mRNA was significantly associated with lymph node metastasis. And this down-regulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 53.4% (62/116) of the TNBC primary breast carcinomas, while it was found in only 24.1% (28/116) of the corresponding nonmalignant tissues. In addition, BRMS1 expression was restored in MDA-MB-231 after treatment with the demethylating agent, 5-aza-2-deoxycytidine (5-Aza-dC), and demethylation of the highly metastatic cells MDA-MB-231 induced invasion suppression of the cells. Furthermore, the suppression of BRMS1 by siRNA transfection enhanced cancer cells invasion. Collectively, our results suggest that the aberrant methylation of BRMS1 frequently occurs in the down-regulation of BRMS1 in TNBC and that it may play a role in the metastasis of breast cancer.

  14. Role of DNA methylation in long-term low-dose γ-rays induced adaptive response in human B lymphoblast cells.

    PubMed

    Ye, Shuang; Yuan, Dexiao; Xie, Yuexia; Pan, Yan; Shao, Chunlin

    2013-11-01

    With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation. HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1-4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured. Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated. Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.

  15. Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

    PubMed Central

    Tennis, Meredith A.; VanScoyk, Michelle M.; Wilson, Lora A.; Kelley, Nicole; Winn, Robert A.

    2012-01-01

    Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC. PMID:22403725

  16. Epigenetic regulation of neural N-glycomics.

    PubMed

    Kizuka, Yasuhiko; Nakano, Miyako; Miura, Yuki; Taniguchi, Naoyuki

    2016-11-01

    Glycan expression is tightly regulated in a cell-type-specific manner, which is essential for the diverse functions of glycans. In particular, neural cells such as neurons and astrocytes are known to express unique functional glycans not found in other cells, and these glycans play critical roles in high-order brain functions and various neurological disorders. However, little is known about how the expression of these neural glycans is established and maintained. Here, we investigated which glycans are expressed in each primary neural cell and how epigenetics contributes to the expression of neural glycans. We first isolated primary neurons, astrocytes, and fibroblasts from mouse embryos and carried out N-glycomic and glycosyltransferase (GlycoT)-transcriptomic analyses to identify N-glycans specific to a particular neural cell type and to clarify the underlying transcriptional basis. We next treated the cells with epigenetic drugs (5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA)) and characterized the changes in GlycoT-transcriptomes and N-glycomes. We found that the N-glycomes in neurons were highly stable and resistant to epigenetic stimulation. In contrast, astrocytes showed dynamic N-glycan changes after treatment, such as a shift in the linkages of sialic acid. These results provide novel insights into how the expression of neural glycans is maintained and epigenetically regulated. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer.

    PubMed

    Kai, Masahiro; Yamamoto, Eiichiro; Sato, Akiko; Yamano, Hiro-O; Niinuma, Takeshi; Kitajima, Hiroshi; Harada, Taku; Aoki, Hironori; Maruyama, Reo; Toyota, Mutsumi; Hatahira, Tomo; Nakase, Hiroshi; Sugai, Tamotsu; Yamashita, Toshiharu; Toyota, Minoru; Suzuki, Hiromu

    2017-02-20

    Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

  18. Aberrant DNA methylation-induced gene inactivation is associated with the diagnosis and/or therapy of T-cell leukemias.

    PubMed

    Kim, Sun Young; Shin, Dong-Yeop; Kim, Sang-Man; Lee, Minyoung; Kim, Eun Ju

    2016-08-01

    Aberrant hypermethylation of tumor suppressor genes is known to play an important role in the development of many tumors, and aberrant DNA hypermethylation was recently identified in hematologic malignancies, where it is thought to hold relevance in leukemogenesis. Here, we report that there are differences in the DNA methylation patterns seen in normal peripheral blood and two T-cell leukemia cell lines. We identify nine genes (CLEC4E, CR1, DBC1, EPO, HAL-DOA, IGF2, IL12B, ITGA1, and LMX1B) that are significantly hypermethylated in T-cell leukemias cell lines, and suggest that aberrant hypermethylation of these normally unmethylated genes may induce their transcriptional and expressional silencing. Furthermore, we observed that the expression levels of DNMT1 and DNMT3a were significantly decreased by 5-aza-2'-deoxycytidine (5-Aza-dC), which is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells and restore the expression levels of their target genes in Jurkat cells. This result suggests that the overexpression of DNMTs could contribute to the development of T-cell leukemias by inducing hypermethylation of the target genes. Together, our results show that aberrant hypermethylation is an important molecular mechanism in the progression of T-cell leukemias, and thus could prove useful as a prognostic and/or diagnostic marker. Moreover, 5-Aza-dC might be a promising candidate for the treatment of T-cell leukemia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Mitochondrial DNA inheritance in the human fungal pathogen Cryptococcus gattii.

    PubMed

    Wang, Zixuan; Wilson, Amanda; Xu, Jianping

    2015-02-01

    The inheritance of mitochondrial DNA (mtDNA) is predominantly uniparental in most sexual eukaryotes. In this study, we examined the mitochondrial inheritance pattern of Cryptococcus gattii, a basidiomycetous yeast responsible for the recent and ongoing outbreak of cryptococcal infections in the US Pacific Northwest and British Columbia (especially Vancouver Island) in Canada. Using molecular markers, we analyzed the inheritance of mtDNA in 14 crosses between strains within and between divergent lineages in C. gattii. Consistent with results from recent studies, our analyses identified significant variations in mtDNA inheritance patterns among strains and crosses, ranging from strictly uniparental to biparental. For two of the crosses that showed uniparental mitochondrial inheritance in standard laboratory conditions, we further investigated the effects of the following environmental variables on mtDNA inheritance: UV exposure, temperature, and treatments with the methylation inhibitor 5-aza-2'-deoxycytidine and with the ubiquitination inhibitor ammonium chloride. Interestingly, one of these crosses showed no response to these environmental variables while the other exhibited diverse patterns ranging from complete uniparental inheritance of the MATa parent mtDNA, to biparental inheritance, and to a significant bias toward inheritance of the MATα parental mtDNA. Our results indicate that mtDNA inheritance in C. gattii differs from that in its closely related species Cryptococcus neoformans.

  20. DNMT1-mediated PTEN hypermethylation confers hepatic stellate cell activation and liver fibrogenesis in rats

    SciTech Connect

    Bian, Er-Bao; Huang, Cheng; Ma, Tao-Tao; Tao, Hui; Zhang, Hui; Cheng, Chang; Lv, Xiong-Wen; Li, Jun

    2012-10-01

    Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation. Highlights: ► PTEN methylation status and loss of PTEN expression ► DNMT1 mediated PTEN hypermethylation. ► Hypermethylation of PTEN contributes to the activation of ERK and AKT pathways.

  1. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

    PubMed

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

  2. Pharmacologic unmasking of epigenetically silenced genes in breast cancer.

    PubMed

    Ostrow, Kimberly Laskie; Park, Hannah Lui; Hoque, Mohammad Obaidul; Kim, Myoung Sook; Liu, Junwei; Argani, Pedram; Westra, William; Van Criekinge, Wim; Sidransky, David

    2009-02-15

    Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2'-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer. Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes. Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P<0.0001, Fisher's exact test). This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.

  3. CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

    SciTech Connect

    Zhang, Heyu; Nan, Xu; Li, Xuefen; Chen, Yan; Zhang, Jianyun; Sun, Lisha; Han, Wenlin; Li, Tiejun

    2014-05-02

    Highlights: • Down-regulation of CMTM5 expression in OSCC tissues was found. • The promoter methylation status of CMTM5 was measured. • CMTM5-v1 inhibited cell proliferation and migration and induced apoptosis. • CMTM5 might act as a putative tumor suppressor gene in OSCC. - Abstract: Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 was down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.

  4. Epigenetic silencing of Na,K-ATPase β 1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma.

    PubMed

    Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

    2014-04-01

    The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β 1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.

  5. Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

    PubMed Central

    Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

    2014-01-01

    The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na+ and uptake of K+ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression. PMID:24452105

  6. DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.

    PubMed

    Stenzig, Justus; Hirt, Marc N; Löser, Alexandra; Bartholdt, Lena M; Hensel, Jan-Tobias; Werner, Tessa R; Riemenschneider, Mona; Indenbirken, Daniela; Guenther, Thomas; Müller, Christian; Hübner, Norbert; Stoll, Monika; Eschenhagen, Thomas

    2016-01-01

    DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 µM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (α-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 µM, nucleosidic inhibitor), RG108 (60 µM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 µM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy.

  7. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.

    PubMed Central

    Herman, J G; Latif, F; Weng, Y; Lerman, M I; Zbar, B; Liu, S; Samid, D; Duan, D S; Gnarra, J R; Linehan, W M

    1994-01-01

    Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers. Images PMID:7937876

  8. DNA methylation of heparanase promoter influences its expression and associated with the progression of human breast cancer.

    PubMed

    Jiao, Fei; Bai, Shi-Yu; Ma, Ying; Yan, Zhong-Hai; Yue, Zhen; Yu, Yuan; Wang, Xin; Wang, Juan

    2014-01-01

    Heparanase promotes tumor invasion and metastasis in several malignancies including breast cancer. However, the roles and regulation mechanisms of heparanase during breast cancer progression are still not fully understood. The aim of this study is to determine the differential regulation of heparanase gene expression in specific stages of breast cancer by DNA methylation. We detected levels of heparanase expression and DNA methylation patterns of its promoter in breast cancer cell lines (MCF-7 and MDA-MB-435) and clinical tissues, respectively. It has been observed that heparanase is highly expressed in the invasive MDA-MB-435 cells with low methylation modification in the heparanase promoter. In contrast, lower expression of heparanase in MCF-7 cells is accompanied by higher methylation in the promoter. Treatment of MCF-7 cells with 5-aza-2'-deoxycytidine (5-aza-dC), a potent demethylating agent, results in induction of heparanase expression and higher invasion potential in vitro and leads to an advantage of tumor formation in vivo. In 54 tissue samples, cancer samples at late stages (stage IV) showed the highest heparanase expression accomplished by little DNA methylation. On the contrary, methylation prevalence is highest in normal tissue and inversely correlated with heparanase expression. A significant correlation between DNA methylation and clinical stage was demonstrated (p = 0.012). Collectively, these results demonstrate that DNA methylation play the regulation role in heparanase gene in different stages of breast cancer and present a direct effect on tumor progression.

  9. An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes

    PubMed Central

    Fagan, Rebecca L.; Wu, Meng; Chédin, Frédéric; Brenner, Charles

    2013-01-01

    DNA methyltransferase 1 (DNMT1) is the enzyme most responsible for epigenetic modification of human DNA and the intended target of approved cancer drugs such as 5-aza-cytidine and 5-aza-2′-deoxycytidine. 5-aza nucleosides have complex mechanisms of action that require incorporation into DNA, and covalent trapping and proteolysis of DNMT isozymes. Direct DNMT inhibitors are needed to refine understanding of the role of specific DNMT isozymes in cancer etiology and, potentially, to improve cancer prevention and treatment. Here, we developed a high throughput pipeline for identification of direct DNMT1 inhibitors. The components of this screen include an activated form of DNMT1, a restriction enzyme-coupled fluorigenic assay performed in 384 well plates with a z-factor of 0.66, a counter screen against the restriction enzyme, a screen to eliminate DNA intercalators, and a differential scanning fluorimetry assay to validate direct binders. Using the Microsource Spectrum collection of 2320 compounds, this screen identified nine compounds with dose responses ranging from 300 nM to 11 µM, representing at least two different pharmacophores with DNMT1 inhibitory activity. Seven of nine inhibitors identified exhibited two to four-fold selectivity for DNMT1 versus DNMT3A. PMID:24236046

  10. Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus

    PubMed Central

    Ito, Yoko; Nativio, Raffaella; Murrell, Adele

    2013-01-01

    Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes. PMID:23585276

  11. Inducible expression of cancer-testis antigens in human prostate cancer

    PubMed Central

    Heninger, Erika; Krueger, Timothy E.G.; Thiede, Stephanie M.; Sperger, Jamie M.; Byers, Brianna L.; Kircher, Madison R.; Kosoff, David; Yang, Bing; Jarrard, David F.; McNeel, Douglas G.; Lang, Joshua M.

    2016-01-01

    Immune tolerance to self-antigens can limit robust anti-tumor immune responses in the use of tumor vaccines. Expression of novel tumor associated antigens can improve immune recognition and lysis of tumor cells. The cancer-testis antigen (CTA) family of proteins has been hypothesized to be an ideal class of antigens due to tumor-restricted expression, a subset of which have been found to induce antibody responses in patients with prostate disease. We demonstrate that CTA expression is highly inducible in five different Prostate Cancer (PC) cell lines using a hypomethylating agent 5-Aza-2′-deoxycytidine (5AZA) and/or a histone deacetylase inhibitor LBH589. These CTAs include NY-ESO1, multiple members of the MAGE and SSX families and NY-SAR35. A subset of CTAs is synergistically induced by the combination of 5AZA and LBH589. We developed an ex vivo organ culture using human PC biopsies for ex vivo drug treatments to evaluate these agents in clinical samples. These assays found significant induction of SSX2 in 9/9 distinct patient samples and NY-SAR35 in 7/9 samples. Further, we identify expression of SSX2 in circulating tumor cells (CTC) from patients with advanced PC. These results indicate that epigenetic modifying agents can induce expression of a broad range of neoantigens in human PC and may serve as a useful adjunctive therapy with novel tumor vaccines and checkpoint inhibitors. PMID:27769045

  12. Overexpression of Ribosomal RNA in the Development of Human Cervical Cancer Is Associated with rDNA Promoter Hypomethylation

    PubMed Central

    Zhou, Hong; Wang, Yapei; Lv, Qiongying; Zhang, Juan; Wang, Qing; Gao, Fei; Hou, Haoli; Zhang, Hao; Zhang, Wei; Li, Lijia

    2016-01-01

    The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer. PMID:27695092

  13. DNA demethylation of the TIM-3 promoter is critical for its stable expression on T cells.

    PubMed

    Chou, F-C; Kuo, C-C; Chen, H-Y; Chen, H-H; Sytwu, H-K

    2016-04-01

    The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is selectively expressed on terminally differentiated T helper 1 (Th1) cells and acts as a negative regulator that terminates Th1 responses. The dysregulation of TIM-3 expression on T cells is associated with several autoimmune phenotypes and with chronic viral infections; however, the mechanism of this regulation is unclear. In this study, we investigated the effect of DNA methylation on the expression of TIM-3. By analyzing the sequences of TIM-3 promoter regions in human and mouse, we identified a CpG island within the TIM-3 promoter and demonstrated that the promoter activity was controlled by DNA methylation. Furthermore, treatment with 5-aza-2'-deoxycytidine enhanced TIM-3 expression on mouse primary CD4(+) T cells under Th0-, Th1- or Th2-polarizing conditions. Finally, pyrosequencing analysis revealed that the methylation level of the TIM-3 promoter gradually decreased after each round of T-cell polarization, and this decrease was inversely correlated with TIM-3 expression. These data suggest that the DNA methylation of the TIM-3 promoter cooperates with lineage-specific transcription factors in the control of Th-cell development. In conclusion, DNA methylation-based regulation of TIM-3 may provide novel insights into understanding the dysregulation of TIM-3 expression under pathogenic conditions.

  14. Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells

    PubMed Central

    TANAKA, TETSUJI; BAI, TAO; TOUJIMA, SAORI; UTSUNOMIYA, TOMOKO; MATSUOKA, TOSHIHIDE; KOBAYASHI, AYA; YAMAMOTO, MADOKA; SASAKI, NORIYUKI; TANIZAKI, YUKO; UTSUNOMIYA, HIROTOSHI; TANAKA, JUNKO; YUKAWA, KAZUNORI

    2012-01-01

    Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2′-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers. PMID:22246465

  15. Differential Promoter Methylation and Histone Modification Contribute to the Brain Specific Expression of the Mouse Mbu-1 Gene

    PubMed Central

    Kim, Byungtak; Kang, Seongeun; Kim, Sun Jung

    2012-01-01

    Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expression to the central nervous system. In this study, to elucidate the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052–0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39–93%. Treatment of 5-aza-2′-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression. PMID:23076708

  16. DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

    PubMed Central

    Cao, Huimin; Wang, Li; Chen, Beibei; Zheng, Peng; He, Yi; Ding, Yubin; Deng, Yushuang; Lu, Xi; Guo, Xiuming; Zhang, Yuping; Li, Yu; Yu, Gang

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the defense against oxidative stress. Cumulative evidence has shown that oxidative stress plays a key role in the pathogenesis of Alzheimer’s disease (AD). Previous animal and clinical studies had observed decreased expression of Nrf2 in AD. However, the underlying regulation mechanisms of Nrf2 in AD remain unclear. Here, we used the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) to test whether Nrf2 expression was regulated by methylation in N2a cells characterizing by expressing human Swedish mutant amyloid precursor protein (N2a/APPswe). We found 5-Aza treatment increased Nrf2 at both messenger RNA and protein levels via downregulating the expression of Dnmts and DNA demethylation. In addition, 5-Aza-mediated upregulation of Nrf2 expression was concomitant with increased nuclear translocation of Nrf2 and higher expression of Nrf2 downstream target gene NAD(P)H:quinone oxidoreductas (NQO1). Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development. PMID:26779013

  17. Cold acclimation alters DNA methylation patterns and confers tolerance to heat and increases growth rate in Brassica rapa

    PubMed Central

    Liu, Tongkun; Li, Ying; Duan, Weike; Huang, Feiyi

    2017-01-01

    Abstract Epigenetic modifications are implicated in plant adaptations to abiotic stresses. Exposure of plants to one stress can induce resistance to other stresses, a process termed cross-adaptation, which is not well understood. In this study, we aimed to unravel the epigenetic basis of elevated heat-tolerance in cold-acclimated Brassica rapa by conducting a genome-wide DNA methylation analysis of leaves from control (CK) and cold-acclimated (CA) plants. We found that both methylation and demethylation occurred during cold acclimation. Two significantly altered pathways, malate dehydrogenase activity and carbon fixation, and 1562 differentially methylated genes, including BramMDH1, BraKAT2, BraSHM4, and Bra4CL2, were identified in CA plants. Genetic validation and treatment of B. rapa with 5-aza-2-deoxycytidine (Aza) suggested that promoter demethylation of four candidate genes increased their transcriptional activities. Physiological analysis suggested that elevated heat-tolerance and high growth rate were closely related to increases in organic acids and photosynthesis, respectively. Functional analyses demonstrated that the candidate gene BramMDH1 (mMDH: mitochondrial malate dehydrogenase) directly enhances organic acids and photosynthesis to increase heat-tolerance and growth rate in Arabidopsis. However, Aza-treated B. rapa, which also has elevated BramMDH1 levels, did not exhibit enhanced heat-tolerance. We therefore suggest that DNA demethylation alone is not sufficient to increase heat-tolerance. This study demonstrates that altered DNA methylation contributes to cross-adaptation. PMID:28158841

  18. HES5 silencing is an early and recurrent change in prostate tumourigenesis

    PubMed Central

    Massie, Charles E; Spiteri, Inmaculada; Ross-Adams, Helen; Luxton, Hayley; Kay, Jonathan; Whitaker, Hayley C; Dunning, Mark J; Lamb, Alastair D; Ramos-Montoya, Antonio; Brewer, Daniel S; Cooper, Colin S; Eeles, Rosalind; Warren, Anne Y; Tavaré, Simon; Neal, David E; Lynch, Andy G

    2015-01-01

    Prostate cancer is the most common cancer in men, resulting in over 10 000 deaths/year in the UK. Sequencing and copy number analysis of primary tumours has revealed heterogeneity within tumours and an absence of recurrent founder mutations, consistent with non-genetic disease initiating events. Using methylation profiling in a series of multi-focal prostate tumours, we identify promoter methylation of the transcription factor HES5 as an early event in prostate tumourigenesis. We confirm that this epigenetic alteration occurs in 86–97% of cases in two independent prostate cancer cohorts (n=49 and n=39 tumour–normal pairs). Treatment of prostate cancer cells with the demethylating agent 5-aza-2′-deoxycytidine increased HES5 expression and downregulated its transcriptional target HES6, consistent with functional silencing of the HES5 gene in prostate cancer. Finally, we identify and test a transcriptional module involving the AR, ERG, HES1 and HES6 and propose a model for the impact of HES5 silencing on tumourigenesis as a starting point for future functional studies. PMID:25560400

  19. Epigenetic genome-wide analysis identifies BEX1 as a candidate tumour suppressor gene in paediatric intracranial ependymoma.

    PubMed

    Karakoula, Katherine; Jacques, Thomas S; Phipps, Kim P; Harkness, William; Thompson, Dominic; Harding, Brian N; Darling, John L; Warr, Tracy J

    2014-04-28

    Promoter hypermethylation and transcriptional silencing is a common epigenetic mechanism of gene inactivation in cancer. To identify targets of epigenetic silencing in paediatric intracranial ependymoma, we used a pharmacological unmasking approach through treatment of 3 ependymoma short-term cell cultures with the demethylating agent 5-Aza-2'-deoxycytidine followed by global expression microarray analysis. We identified 55 candidate epigenetically silenced genes, which are involved in the regulation of apoptosis, Wnt signalling, p53 and cell differentiation. The methylation status of 26 of these genes was further determined by combined bisulfite restriction analysis (COBRA) and genomic sequencing in a cohort of 40 ependymoma samples. The most frequently methylated genes were BEX1 (27/40 cases), BAI2 (20/40), CCND2 (18/40), and CDKN2A (14/40). A high correlation between promoter hypermethylation and decreased gene expression levels was established by real-time quantitative PCR, suggesting the involvement of these genes in ependymoma tumourigenesis. Furthermore, ectopic expression of brain-expressed X-linked 1 (BEX1) in paediatric ependymoma short-term cell cultures significantly suppressed cell proliferation and colony formation. These data suggest that promoter hypermethylation contributes to silencing of target genes in paediatric intracranial ependymoma. Epigenetic inactivation of BEX1 supports its role as a candidate tumour suppressor gene in intracranial ependymoma, and a potential target for novel therapies for ependymoma in children.

  20. Methylation subtypes and large-scale epigenetic alterations in gastric cancer.

    PubMed

    Zouridis, Hermioni; Deng, Niantao; Ivanova, Tatiana; Zhu, Yansong; Wong, Bernice; Huang, Dan; Wu, Yong Hui; Wu, Yingting; Tan, Iain Beehuat; Liem, Natalia; Gopalakrishnan, Veena; Luo, Qin; Wu, Jeanie; Lee, Minghui; Yong, Wei Peng; Goh, Liang Kee; Teh, Bin Tean; Rozen, Steve; Tan, Patrick

    2012-10-17

    Epigenetic alterations are fundamental hallmarks of cancer genomes. We surveyed the landscape of DNA methylation alterations in gastric cancer by analyzing genome-wide CG dinucleotide (CpG) methylation profiles of 240 gastric cancers (203 tumors and 37 cell lines) and 94 matched normal gastric tissues. Cancer-specific epigenetic alterations were observed in 44% of CpGs, comprising both tumor hyper- and hypomethylation. Twenty-five percent of the methylation alterations were significantly associated with changes in tumor gene expression. Whereas most methylation-expression correlations were negative, several positively correlated methylation-expression interactions were also observed, associated with CpG sites exhibiting atypical transcription start site distances and gene body localization. Methylation clustering of the tumors revealed a CpG island methylator phenotype (CIMP) subgroup associated with widespread hypermethylation, young patient age, and adverse patient outcome in a disease stage-independent manner. CIMP cell lines displayed sensitivity to 5-aza-2'-deoxycytidine, a clinically approved demethylating drug. We also identified long-range regions of epigenetic silencing (LRESs) in CIMP tumors. Combined analysis of the methylation, gene expression, and drug treatment data suggests that certain LRESs may silence specific genes within the region, rather than all genes. Finally, we discovered regions of long-range tumor hypomethylation, associated with increased chromosomal instability. Our results provide insights into the epigenetic impact of environmental and biological agents on gastric epithelial cells, which may contribute to cancer.

  1. High incidence of LRAT promoter hypermethylation in colorectal cancer correlates with tumor stage

    PubMed Central

    Pincas, Hanna; Huang, Jianmin; Zachariah, Emmanuel; Zeng, Zhaoshi; Notterman, Daniel A.; Paty, Philip; Barany, Francis

    2015-01-01

    Lecithin:retinol acyltransferase (LRAT) is a major enzyme involved in vitamin A/retinol metabolism, which regulates various physiological processes like cell proliferation and differentiation. LRAT expression is reduced in numerous cancers, yet the underlying mechanisms have remained undefined. We hypothesized that methylation silencing may contribute to decreased LRAT gene expression in colorectal cancer (CRC). LRAT hypermethylation status was analyzed in five CRC cell lines, 167 colorectal tumors, and 69 adjacent normal colonic mucosae, using a quantitative bisulfite/PCR/LDR/Universal Array assay. LRAT transcription levels were determined by real-time RT-PCR in a subset of tumors and matched normal tissues and in CRC cell lines that were treated with a demethylating agent, 5-aza-2′-deoxycytidine. The incidence of LRAT hypermethylation was significantly higher in colorectal tumors than in adjacent normal mucosae (p = 0.0025). Aberrant methylation occurred in 51 % of microsatellite-stable CRCs, in 84 % of microsatellite-unstable CRCs, and in 12 out of 13 colonic polyps. The number of hypermethylated LRAT events was inversely correlated with CRC stage (p < 0.0001). Importantly, LRAT hypermethylation was associated with decreased mRNA level in CRC clinical specimens, and demethylation treatment resulted in LRAT transcriptional reactivation. Our data support the idea that LRAT promoter hypermethylation associates with LRAT gene expression in CRC. The higher frequency of LRAT hypermethylation in colonic polyps and early-stage CRCs indicates that it may occur early in malignant progression. PMID:25260806

  2. Frequent epigenetic inactivation of KIBRA, an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network, is associated with specific genetic event in B-cell acute lymphocytic leukemia.

    PubMed

    Hill, Victoria K; Dunwell, Thomas L; Catchpoole, Daniel; Krex, Dietmar; Brini, Anna T; Griffiths, Mike; Craddock, Charles; Maher, Eamonn R; Latif, Farida

    2011-03-01

    The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in < 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 [t(12;21) (p13;q22)] chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.

  3. Tissue Inhibitor of Metalloproteinase 1 Expression Associated with Gene Demethylation Confers Anoikis Resistance in Early Phases of Melanocyte Malignant Transformation1

    PubMed Central

    Ricca, Tatiana I; Liang, Gangning; Suenaga, Ana Paula M; Han, Sang W; Jones, Peter A; Jasiulionis, Miriam G

    2009-01-01

    Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2′-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation. PMID:19956395

  4. Combined analysis of DNA methylation and cell cycle in cancer cells.

    PubMed

    Desjobert, Cécile; El Maï, Mounir; Gérard-Hirne, Tom; Guianvarc'h, Dominique; Carrier, Arnaud; Pottier, Cyrielle; Arimondo, Paola B; Riond, Joëlle

    2015-01-01

    DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.

  5. Frequent silencing of protocadherin 17, a candidate tumour suppressor for esophageal squamous cell carcinoma.

    PubMed

    Haruki, Shigeo; Imoto, Issei; Kozaki, Ken-ichi; Matsui, Takeshi; Kawachi, Hiroshi; Komatsu, Shuhei; Muramatsu, Tomoki; Shimada, Yutaka; Kawano, Tatsuyuki; Inazawa, Johji

    2010-06-01

    Protocadherins are a subfamily of the cadherin superfamily, but little is known about their functions. We identified a homozygous loss of protocadherin (PCDH) 17 in the course of a program to screen a panel of esophageal squamous cell carcinoma (ESCC) cell lines for genomic copy number aberrations. PCDH17 messenger RNA was expressed in normal esophageal tissue but not in the majority of ESCC cell lines without a homozygous deletion of this gene and restored in gene-silenced ESCC cells after treatment with 5-aza-2'-deoxycytidine. The DNA methylation status of the PCDH17 CpG island correlated inversely with the PCDH17 expression, and a putative methylation target region showed promoter activity. The methylation of the PCDH17 promoter was also associated with the silencing of gene expression in primary ESCC partly. Among primary ESCC cases, the silencing of PCDH17 protein expression was associated with a poorer differentiation status of ESCC cells and possibly with prognosis in a subset of this tumour. Restoration of PCDH17 expression in ESCC cells reduced cell proliferation and migration/invasion. These results suggest that silencing of PCDH17 expression through hypermethylation of the promoter or other mechanisms leads to loss of its tumour-suppressive activity, which may be a factor in the carcinogenesis of a subgroup of ESCCs.

  6. Identification of Methylation-Driven, Differentially Expressed STXBP6 as a Novel Biomarker in Lung Adenocarcinoma

    PubMed Central

    Lenka, Govinda; Tsai, Mong-Hsun; Lin, Hsin-Chieh; Hsiao, Jen-Hao; Lee, Yi-Ching; Lu, Tzu-Pin; Lee, Jang-Ming; Hsu, Chung-Ping; Lai, Liang-Chuan; Chuang, Eric Y.

    2017-01-01

    DNA methylation is an essential epigenetic marker associated with the silencing of gene expression. Although various genome-wide studies revealed aberrantly methylated gene targets as molecular biomarkers for early detection, the survival rate of lung cancer patients is still poor. In order to identify methylation-driven biomarkers, genome-wide changes in DNA methylation and differential expression in 32 pairs of lung adenocarcinoma and adjacent normal lung tissue in non-smoking women were examined. This concurrent analysis identified 21 negatively correlated probes (r ≤ −0.5), corresponding to 17 genes. Examining the endogenous expression in lung cancer cell lines, five of the genes were found to be significantly down-regulated. Furthermore, in tumor cells alone, 5-aza-2′-deoxycytidine treatment increased the expression levels of STXBP6 in a dose dependent manner and pyrosequencing showed higher percentage of methylation in STXBP6 promoter. Functional analysis revealed that overexpressed STXBP6 in A549 and H1299 cells significantly decreased cell proliferation, colony formation, and migration, and increased apoptosis. Finally, significantly lower survival rates (P < 0.05) were observed when expression levels of STXBP6 were low. Our results provide a basis for the genetic etiology of lung adenocarcinoma by demonstrating the possible role of hypermethylation of STXBP6 in poor clinical outcomes in lung cancer patients. PMID:28198450

  7. Environment factors can influence mitochondrial inheritance in the fungus Cryptococcus neoformans.

    PubMed

    Yan, Zhun; Sun, Sheng; Shahid, Mori; Xu, Jianping

    2007-05-01

    Cryptococcus neoformans is a model basidiomycete yeast. Strains of this species belong to one of two mating types: mating type a (MATa) or mating type alpha (MATalpha). In typical crosses between MATa and MATalpha strains, the progeny inherit mitochondria from the MATa parent. However, the underlying mechanisms remain largely unknown. To help elucidate the molecular mechanisms, we examined the effects of four environmental factors on the patterns of mtDNA inheritance. These factors are temperature, UV irradiation, and the addition of either the methylation inhibitor 5-aza-2'-deoxycytidine (5-adc) or the ubiquitination inhibitor ammonium chloride. Except temperature, the other three factors have been shown to influence organelle inheritance during sexual mating in other eukaryotes. Our results indicate that while the application of 5-adc or ammonium chloride did not influence mtDNA inheritance in C. neoformans, both UV irradiation and high temperature treatments did. Progeny from a cross involving a high temperature-sensitive mutant with the calcineurin subunit A gene deleted showed biparental mtDNA inheritance in all examined temperatures, consistent with a role of calcineurin and temperature in mtDNA inheritance. Furthermore, the zygote progeny population from a cross performed at a high-temperature environment had a greater variability in their vegetative fitness than that from the same cross conducted at a low temperature. Our results indicate a potentially adaptive role of biparental mtDNA inheritance and mtDNA recombination in certain environments in C. neoformans.

  8. Inhibition of DNA Methylation Impairs Synaptic Plasticity during an Early Time Window in Rats

    PubMed Central

    Díaz, Paula; Ardiles, Álvaro O.

    2016-01-01

    Although the importance of DNA methylation-dependent gene expression to neuronal plasticity is well established, the dynamics of methylation and demethylation during the induction and expression of synaptic plasticity have not been explored. Here, we combined electrophysiological, pharmacological, molecular, and immunohistochemical approaches to examine the contribution of DNA methylation and the phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) to synaptic plasticity. We found that, at twenty minutes after theta burst stimulation (TBS), the DNA methylation inhibitor 5-aza-2-deoxycytidine (5AZA) impaired hippocampal long-term potentiation (LTP). Surprisingly, after two hours of TBS, when LTP had become a transcription-dependent process, 5AZA treatment had no effect. By comparing these results to those in naive slices, we found that, at two hours after TBS, an intergenic region of the RLN gene was hypomethylated and that the phosphorylation of residue S80 of MeCP2 was decreased, while the phosphorylation of residue S421 was increased. As expected, 5AZA affected only the methylation of the RLN gene and exerted no effect on MeCP2 phosphorylation patterns. In summary, our data suggest that tetanic stimulation induces critical changes in synaptic plasticity that affects both DNA methylation and the phosphorylation of MeCP2. These data also suggest that early alterations in DNA methylation are sufficient to impair the full expression of LTP. PMID:27493805

  9. Ex vivo modulation of response to prednisolone in childhood acute lymphoblastic leukaemia.

    PubMed

    Styczynski, Jan; Wysocki, Mariusz

    2006-05-01

    We hypothesised that the intensity of mechanisms of glucocorticoid resistance in childhood acute lymphoblastic leukaemia might be decreased by concurrent ex vivo use of compounds with specific blocking or activating properties at different steps of the glucocorticoid intracellular pathway. The following modifiers were used: ciclosporin A, rifampicin, doxycycline, meta-iodobenzylguanidine, buthionine sulfoximine, ethacrinic acid, pentoxifylline, indomethacin, rotenone, forskolin, olomoucin, 5-aza-2'-deoxycytidine, 3-aminobenzamide, O(6)-benzylguanidine and nitroprusside sodium. All modulators sensitised lymphoblasts and potentiated prednisolone cytotoxicity in most cases indicating that various compounds, which can influence the antileukaemic effect of prednisolone during anticancer therapy, might modulate some mechanisms of glucocorticoid resistance.

  10. The sugar ring of the nucleoside is required for productive substrate positioning in the active site of human deoxycytidine kinase (dCK): implications for the development of dCK-activated acyclic guanine analogs

    PubMed Central

    Hazra, Saugata; Konrad, Manfred; Lavie, Arnon

    2010-01-01

    The low toxicity of acyclovir (ACV) is mainly due to the fact that human nucleoside kinases have undetectable phosphorylation rates with this acyclic guanine analog. In contrast, herpes virus thymidine kinase (HSV1-TK) readily activates ACV. We wanted to understand why human deoxycytidine kinase (dCK), which is related to HSV1-TK and phosphorylates deoxyguanosine, does not accept acyclic guanine analogs as substrates. Therefore, we crystallized dCK in complex with ACV at the nucleoside phosphoryl acceptor site, and UDP at the phosphoryl donor site. The structure reveals that while ACV does bind at the dCK active site, it does so adopting a non-productive conformation. Despite binding ACV, the enzyme remains in the open, inactive state. In comparison to ACV binding to HSV1-TK, in dCK the nucleoside base adopts a different orientation related by about a 60 degree rotation. Our analysis suggests that dCK would phosphorylate acyclic guanine analogs if they can induce a similar rotation. PMID:20684612

  11. Successful treatment of recurrent vulvar intraepithelial neoplasia resistant to interferon and isotretinoin with cidofovir.

    PubMed

    Koonsaeng, S; Verschraegen, C; Freedman, R; Bossens, M; Kudelka, A; Kavanagh, J; Sittisomwong, T; DeClercq, E; Snoeck, R

    2001-06-01

    Vulvar intraepithelial neoplasias are difficult to eradicate completely without extensive surgical intervention. Cidofovir, a deoxycytidine monophosphate analog, may have a therapeutic role in this disease. A 43-year-old woman with a 20-year history of genital warts presented with extensive vulvar intraepithelial neoplasia III, and refused surgical resection. Topical cidofovir 1% in Beeler base completely eradicated the lesion. Successive treatment applications, however, were necessary. Cidofovir is a promising topical antiviral compound for HPV induced vulvar intraepithelial neoplasia. Copyright 2001 Wiley-Liss, Inc.

  12. Direct transfection of miR-137 mimics is more effective than DNA demethylation of miR-137 promoter to augment anti-tumor mechanisms of delphinidin in human glioblastoma U87MG and LN18 cells.

    PubMed

    Chakrabarti, Mrinmay; Ray, Swapan K

    2015-11-15

    Glioblastoma is the deadliest brain tumor in humans. Recent studies suggested that 5-aza-2-deoxycytidine (AzaC) could inhibit cell cycle progression in human glioblastoma stem cells by an indirect increase in expression of the tumor suppressor microRNA-137 (miR-137). Delphinidin (DPN), a new anthocyanidin, inhibits cell growth in different cancers. We investigated inhibition of glioblastoma growth after indirect or direct overexpression of miR-137 and then DPN treatment. The highest inhibition of cell growth occurred due to treatment with combination of 10 μM AzaC and 50 μM DPN in human glioblastoma U87MG and LN18 cells. The methylation sensitive-polymerase chain reaction (MS-PCR) results showed that AzaC inhibited methylation of miR-137 promoter region, which was hypermethylated in both glioblastoma cell lines, to cause indirect increase in miR-137 expression. Our results also indicated the highest miR-137 expression after direct transfection of miR-137 mimics and DPN treatment. Combination of miR-137 mimics transfection and DPN treatment caused the highest inhibition of cell invasion and prevented angiogenic network formation due to the least expression of angiogenic factor (VEGF) in human glioblastoma cells in co-culture with human microvascular endothelial cells. This combination strategy most effectively inhibited survival factors (p-Akt and NF-κB), angiogenic factors (VEGF and b-FGF), growth factor receptor (EGFR), and invasive factors (MMP-9 and MMP-2). Direct overexpression of miR-137 most effectively augmented efficacy of DPN to induce apoptosis with activation of extrinsic and intrinsic pathways. So, sequential miR-137 overexpression and DPN treatment could be a promising combination treatment to inhibit growth of human glioblastoma cells.

  13. Renal tumours in a Tsc1+/- mouse model show epigenetic suppression of organic cation transporters Slc22a1, Slc22a2 and Slc22a3, and do not respond to metformin.

    PubMed

    Yang, Jian; Kalogerou, Maria; Gallacher, John; Sampson, Julian R; Shen, Ming Hong

    2013-04-01

    Metformin, a substrate of several poly-specific organic cation transporters, is a widely used biguanide for the treatment of type II diabetes. Recent studies suggest that metformin attenuates mTORC1 signalling by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) in the presence or absence of a functional hamartin/tuberin (TSC1/TSC2) complex. Metformin has also been reported to inhibit mTORC1 independent of AMPK through p53-dependent regulated in development and DNA damage responses 1 (REDD1) or by inhibiting Rag GTPases. These observations suggest that metformin could have therapeutic potential for tuberous sclerosis, an inherited disorder characterised by the aberrant activation of mTORC1 and the development of tumours in many organs, including the kidneys. In this study, we investigated the effect of metformin on renal lesions in a Tsc1(+/-) mouse model of tuberous sclerosis. Continuous treatment of metformin for 9 months at doses of up to 600 mg/kg/day had no significant effect on renal lesions in nine treated mice compared to 10 controls. Metformin treatment appeared to attenuate mTORC1 signalling in Tsc1(+/-) kidney tissues but not in renal tumours. Surprisingly, the expression of the organic cation transporters Slc22a1, Slc22a2 and Slc22a3 essential for the cellular uptake of metformin was highly suppressed in renal tumours. Treatment of cultured cells derived from a Tsc1-associated renal tumour with 5-aza-2-deoxycytidine or trichostatin A greatly increased the expression of these genes. These data suggest that the epigenetic suppression of the organic cation transporters in Tsc-associated mouse renal tumours may contribute to the lack of response to metformin treatment.

  14. Role of DNA methylation in expression control of the IKZF3-GSDMA region in human epithelial cells.

    PubMed

    Moussette, Sanny; Al Tuwaijri, Abeer; Kohan-Ghadr, Hamid-Reza; Elzein, Samar; Farias, Raquel; Bérubé, Julie; Ho, Bianca; Laprise, Catherine; Goodyer, Cynthia G; Rousseau, Simon; Naumova, Anna K

    2017-01-01

    Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site's methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8-13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation.

  15. Role of DNA methylation in expression control of the IKZF3-GSDMA region in human epithelial cells

    PubMed Central

    Kohan-Ghadr, Hamid-Reza; Elzein, Samar; Farias, Raquel; Bérubé, Julie; Ho, Bianca; Laprise, Catherine; Goodyer, Cynthia G.; Rousseau, Simon

    2017-01-01

    Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site’s methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8–13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation. PMID:28241063

  16. Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

    PubMed

    Raghavan, Vijay; Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Yamada, Masayoshi; Morisada, Megan; Labhasetwar, Vinod

    2015-10-27

    Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport

  17. Epigenetic regulation of macrophage polarization and inflammation by DNA methylation in obesity

    PubMed Central

    Wang, Xianfeng; Yu, Liqing; Shi, Huidong

    2016-01-01

    Obesity is associated with increased classically activated M1 adipose tissue macrophages (ATMs) and decreased alternatively activated M2 ATMs, both of which contribute to obesity-induced inflammation and insulin resistance. However, the underlying mechanism remains unclear. We find that inhibiting DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNA methyltransferase 1 (DNMT1) deletion promotes alternative activation and suppresses inflammation in macrophages. Consistently, mice with myeloid DNMT1 deficiency exhibit enhanced macrophage alternative activation, suppressed macrophage inflammation, and are protected from obesity-induced inflammation and insulin resistance. The promoter and 5′-untranslated region of peroxisome proliferator-activated receptor γ1 (PPARγ1) are enriched with CpGs and are epigenetically regulated. The saturated fatty acids stearate and palmitate and the inflammatory cytokine TNF-α significantly increase, whereas the TH2 cytokine IL-4 significantly decreases PPARγ1 promoter DNA methylation. Accordingly, inhibiting PPARγ1 promoter DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNMT1 deletion promotes macrophage alternative activation. Our data therefore establish DNA hypermethylation at the PPARγ1 promoter induced by obesity-related factors as a critical determinant of ATM proinflammatory activation and inflammation, which contributes to insulin resistance in obesity. PMID:27882346

  18. TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells

    PubMed Central

    Yamamoto, Eiichiro; Harada, Taku; Aoki, Hironori; Maruyama, Reo; Toyota, Mutsumi; Sasaki, Yasushi; Sugai, Tamotsu; Tokino, Takashi; Nakase, Hiroshi

    2016-01-01

    Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2’-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2’-deoxycytidine in CRC cells. PMID:27977763

  19. Epigenetic regulation of macrophage polarization and inflammation by DNA methylation in obesity.

    PubMed

    Wang, Xianfeng; Cao, Qiang; Yu, Liqing; Shi, Huidong; Xue, Bingzhong; Shi, Hang

    2016-11-17

    Obesity is associated with increased classically activated M1 adipose tissue macrophages (ATMs) and decreased alternatively activated M2 ATMs, both of which contribute to obesity-induced inflammation and insulin resistance. However, the underlying mechanism remains unclear. We find that inhibiting DNA methylation pharmacologically using 5-aza-2'-deoxycytidine or genetically by DNA methyltransferase 1 (DNMT1) deletion promotes alternative activation and suppresses inflammation in macrophages. Consistently, mice with myeloid DNMT1 deficiency exhibit enhanced macrophage alternative activation, suppressed macrophage inflammation, and are protected from obesity-induced inflammation and insulin resistance. The promoter and 5'-untranslated region of peroxisome proliferator-activated receptor γ1 (PPARγ1) are enriched with CpGs and are epigenetically regulated. The saturated fatty acids stearate and palmitate and the inflammatory cytokine TNF-α significantly increase, whereas the TH2 cytokine IL-4 significantly decreases PPARγ1 promoter DNA methylation. Accordingly, inhibiting PPARγ1 promoter DNA methylation pharmacologically using 5-aza-2'-deoxycytidine or genetically by DNMT1 deletion promotes macrophage alternative activation. Our data therefore establish DNA hypermethylation at the PPARγ1 promoter induced by obesity-related factors as a critical determinant of ATM proinflammatory activation and inflammation, which contributes to insulin resistance in obesity.

  20. Changes in global gene expression in response to chemical and genetic perturbation of chromatin structure

    USDA-ARS?s Scientific Manuscript database

    DNA methylation and histone acetylation are important for controlling gene expression in all eukaryotes. Microarray analysis revealed an altered gene expression profile after treatment with the DNA methylation inhibitor 5-aza-2’ deoxyctidine (5-AC), which included the upregulation of many transposab...

  1. Cell cycle regulation of human endometrial stromal cells during decidualization.

    PubMed

    Logan, Philip C; Steiner, Michael; Ponnampalam, Anna P; Mitchell, Murray D

    2012-08-01

    Differentiation of endometrial stromal cells into decidual cells is crucial for optimal endometrial receptivity. Data from our previous microarray study implied that expression of many cell cycle regulators are changed during decidualization and inhibition of DNA methylation in vitro. In this study, we hypothesized that both the classic progestin treatment and DNA methylation inhibition would inhibit stromal cell proliferation and cell cycle transition. The human endometrial stromal cell line (HESC) was treated from 2 days to 18 days with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (AZA), a mixture of estradiol/progestin/cyclic adenosine monophosphate ([cAMP]; medroxy-progesterone acetate [MPA mix]) or both. Cell growth was measured by cell counting, cell cycle transition and apoptosis were analyzed by flow cytometry, expression of cell cycle regulators were analyzed by quantitative polymerase chain reaction (qPCR) and Western blotting, and change in DNA methylation profiles were detected by methylation-specific PCR. Both AZA and MPA mix inhibited the proliferation of HESC for at least 7 days. Treatment with MPA mix resulted in an early G0/G1 inhibition followed by G2/M phase inhibition at 18 days. In contrast, AZA treatment inhibited cell cycle progression at the G2/M phase throughout. The protein levels of p21(Cip1)and 14-3-3σ were increased with both AZA and MPA mix treatments without any change in the DNA methylation profiles of the genes. Our data imply that the decidualization of HESC is associated with cell cycle arrest at G0/G1 phase initially and G2/M phase at later stages. Our results also suggest that p53 pathway members play a role in the cell cycle regulation of endometrial stromal cells.

  2. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in adenomyosis and its rectification by a histone deacetylase inhibitor and a demethylation agent.

    PubMed

    Jichan Nie; Xishi Liu; Guo, Sun-Wei

    2010-11-01

    Adenomyosis is a fairly common gynecologic disease with unknown pathogenesis. We sought to investigate as to whether the promoter of progesterone receptor isoform B (PR-B) is hypermethylated in adenomyosis and to investigate the treatment of ectopic endometrial stromal cells with trichostatin A (TSA), a histone deacetylase inhibitor (HDI), and 5-aza-2-deoxycytidine (ADC), a demethylation agent, on PR-B gene and protein expression, and on cell viability. Ectopic endometrial tissue specimens were obtained from 9 women with adenomyosis whereas control endometrial tissue samples were obtained from 8 women with surgically diagnosed benign ovarian cysts but without any clinical history of endometriosis/adenomyosis/ myoma. Endometrial stromal cells were isolated, purified, cultured, and analyzed by methylation-specific polymerase chain reaction (PCR), real-time reverse transcriptase PCR (RT-PCR), and Western blot analysis, cell viability assays, and fluorescence-activated cell sorting. We found that none of the normal endometrial stromal cells had PR-B promoter methylation. In contrast, 2 out of 3 ectopic endometrial stromall cells had PR-B hypermethylation (P < .05). The treatment with both TSA and ADC elevated PR-B gene and protein expression in ectopic, but not in normal, endometrial stromal cells. Both TSA and ADC treatment dose-dependently reduced cell viability of ectopic endometrial stromal cells. Trichostatin A and ADC treatment also suppressed the cell cycle progression in ectopic endometrial stromal cells. Thus, this study provides the first piece of evidence that adenomyosis has epigenetic aberration and may also be an epigenetic disease amenable to rectification by pharmacological means. This perspective may shed new light onto the pathogenesis of adenomyosis and lead to novel ways to treat the disease.

  3. Alteration of transcriptional profile in human bronchial epithelial cells induced by cigarette smoke condensate.

    PubMed

    Hu, Ying-Chun; Yang, Zhi-Hua; Zhong, Ke-Jun; Niu, Li-Jing; Pan, Xiu-Jie; Wu, De-Chang; Sun, Xian-Jun; Zhou, Ping-Kun; Zhu, Mao-Xiang; Huo, Yan-Ying

    2009-10-08

    Despite the significance of cigarette smoke for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of human cancers are not well-understood. In our present study, the oncogenic transforming effects of cigarette smoke condensate (CSC) were examined using papillomavirus-immortalized human bronchial epithelial cells (BEP2D). Growth kinetics, saturation density, resistance to serum-induced terminal differentiation, anchorage-independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in BEP2D cells. Illumina microarray platforms were used to explore the CSC-induced alteration of global mRNA expression profiles of the earlier period and the advanced stage of CSC-treated BEP2D cells. We showed here that a series of sequential steps arose among CSC-treated immortalized human bronchial epithelial cells, including altered growth kinetics, resistance to serum-induced terminal differentiation, and anchorage-independence growth. In the earlier period of CSC treatment, 265 genes were down-regulated and 63 genes were up-regulated, respectively, and in the advanced stage of CSC treatment, 313 genes were down-regulated and 145 genes were up-regulated, respectively. Notably, among those genes, the expression of some of imprinted genes such as IGF2, NDN, H19 and MEG3 were all silenced or down-regulated in CSC-treated cells. These genes reactivated after 5 microM 5-aza-2-deoxycytidine (5-aza-dC) treatment. These results demonstrated that long-term treatment of human bronchial epithelial cells with CSC may adversely affect their genetic and epigenetic integrity and lead to further transformation.

  4. DAC can restore expression of NALP1 to suppress tumor growth in colon cancer.

    PubMed

    Chen, C; Wang, B; Sun, J; Na, H; Chen, Z; Zhu, Z; Yan, L; Ren, S; Zuo, Y

    2015-01-22

    Despite recent progress in the identification of genetic and molecular alternations in colorectal carcinoma, the precise molecular pathogenesis remains unclear. NALP1 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 1) is a member of the nucleotide-binding oligomerization domain-like receptor family of proteins that are key organization proteins in the inflammasome. It is reported that NALP1 plays a central role in cell apoptosis, pyroptosis, inflammatory reactions and autoimmune diseases. DAC (5-aza-2-deoxycytidine) is an antitumor drug useful to lung cancer, myelodysplastic disorders, myelodysplasia and acute myeloid leukemia. In this study, we examined the expression of NALP1 in human normal and cancerous colon tissues using tissue microarray, western blot and quantitative real-time PCR and we measured the expression of NALP1 in three kinds of colon cancer cell lines and animal models before and after treatment with DAC. Furthermore, we examined the treatment effects of DAC on colon cancer in our animal model. Our data indicate that NALP1 is expressed low in human colorectal tumoral tissues relative to paratumoral tissues and was associated with the survival and tumor metastasis of patients. The expression of NALP1 increased after treatment with DAC both in vitro and in vivo. Furthermore, DAC suppressed the growth of colon cancer and increased lifespan in mouse model. Therefore, we conclude that NALP1 is expressed low in colon cancer and associated with the survival and tumor metastasis of patients, and treatment with DAC can restore NALP1 levels to suppress the growth of colon cancer.

  5. Epigenetic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity.

    PubMed

    Cantor, Joshua P; Iliopoulos, Dimitrios; Rao, Atul S; Druck, Teresa; Semba, Shuho; Han, Shuang-Yin; McCorkell, Kelly A; Lakshman, Thiru V; Collins, Joshua E; Wachsberger, Phyllis; Friedberg, Joseph S; Huebner, Kay

    2007-01-01

    Epigenetic changes involved in cancer development, unlike genetic changes, are reversible. DNA methyltransferase and histone deacetylase inhibitors show antiproliferative effects in vitro, through tumor suppressor reactivation and induction of apoptosis. Such inhibitors have shown activity in the treatment of hematologic disorders but there is little data concerning their effectiveness in treatment of solid tumors. FHIT, WWOX and other tumor suppressor genes are frequently epigenetically inactivated in lung cancers. Lung cancer cell clones carrying conditional FHIT or WWOX transgenes showed significant suppression of xenograft tumor growth after induction of expression of the FHIT or WWOX transgene, suggesting that treatments to restore endogenous Fhit and Wwox expression in lung cancers would result in decreased tumorigenicity. H1299 lung cancer cells, lacking Fhit, Wwox, p16(INK4a) and Rassf1a expression due to epigenetic modifications, were used to assess efficacy of epigenetically targeted protocols in suppressing growth of lung tumors, by injection of 5-aza-2-deoxycytidine (AZA) and trichostatin A (TSA) in nude mice with established H1299 tumors. High doses of intraperitoneal AZA/TSA suppressed growth of small tumors but did not affect large tumors (200 mm(3)); lower AZA doses, administered intraperitoneally or intratumorally, suppressed growth of small tumors without apparent toxicity. Responding tumors showed restoration of Fhit, Wwox, p16(INKa), Rassf1a expression, low mitotic activity, high apoptotic fraction and activation of caspase 3. These preclinical studies show the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of re-expressed tumor suppressors as markers and effectors of the responses.

  6. Deregulation of Wnt/β-catenin signaling through genetic or epigenetic alterations in human neuroendocrine tumors

    PubMed Central

    Evers, B.Mark

    2013-01-01

    Carcinoid tumors are rare neuroendocrine tumors (NETs) that are increasing in incidence. Mutation and altered expression of Wnt/β-catenin signaling components have been described in many tumors but have not been well-studied in NETs. Here, we observed accumulation of β-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutational analysis, the mutations of β-catenin (I35S) and APC (E1317Q, T1493T) were identified in NET cells and the tissues. Expression of representative Wnt inhibitors was absent or markedly decreased in BON, a human pancreatic carcinoid cell line; treatment with 5-aza-2′-deoxycytidine (5-aza-CdR) increased expression levels of the Wnt inhibitors. Methylation analyses demonstrated that CpG islands of SFRP-1 and Axin-2 were methylated, whereas the promoters of DKK-1, DKK-3 and WIF-1 were unmethylated in four NET cells. Aberrant methylation of SFRP-1 was particularly observed in most of clinical NET tissues. In addition, the repression of these unmethylated genes was associated with histone H3 lysine 9 dimethylation (H3K9me2) in BON cells. Together, 5-aza-CdR treatment inhibited cell proliferation and decreased the protein levels of H3K9me2 and G9a. Moreover, a novel G9a inhibitor, UNC0638, suppressed BON cell proliferation through inhibition of Wnt/β-catenin pathway. Overexpression of the inhibitory genes, particularly SFRP-1 and WIF-1 in BON cells, resulted in suppression of anchorage-independent growth and inhibition of tumor growth in mice. Our findings suggest that aberrant Wnt/β-catenin signaling, through either mutations or epigenetic silencing of Wnt antagonists, contributes to the pathogenesis and growth of NETs and have important clinical implications for the prognosis and treatment of NETs. PMID:23354304

  7. Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

    PubMed

    Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

    2016-08-01

    Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. Copyright © 2016 by The American Society for Pharmacology and

  8. Effects of DNA methylation inhibitors and conventional antidepressants on mice behaviour and brain DNA methylation levels.

    PubMed

    Sales, Amanda Juliana; Joca, Sâmia Regiane Lourenço

    2016-02-01

    Stress increases DNA methylation and decreases the expression of genes involved in neural plasticity, while treatment with DNA methyltransferase inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in preclinical models. Therefore, the aim of the present work was to further investigate the potential antidepressant-like effect induced by DNMTi by evaluating the behavioural effects induced by associating DNMTi treatment with conventional antidepressant drugs in mice submitted to the forced swimming test (FST). In addition, brain levels of DNA methylation were also investigated. Mice received systemic injections of 5-aza-2'-deoxycytidine (5-AzaD, 0.1, 0.2 mg/kg), RG108 (0.1, 0.2, 0.4 mg/kg), desipramine (DES, 2.5, 5, 10 mg/kg) or fluoxetine (FLX, 5, 10, 20, 30 mg/kg) and were submitted to the FST or to the open field test (OFT). Additional groups received a combination of subeffective doses of 5-AzaD or RG108 (DNMTi) with subeffective doses of DES or FLX (antidepressants). Subeffective doses of RG108 (0.1 mg/kg) or 5-AzaD (0.1 mg/kg) in association with subeffective doses of DES (2.5 mg/kg) or FLX (10 mg/kg) induced significant antidepressant-like effects. Effective doses of RG108 (0.2 mg/kg), 5-AzaD (0.2 mg/kg), DES (10 mg/kg) and FLX (20 mg/kg) atenuated stress-induced changes in DNA methylation levels in the hippocampus and prefrontal cortex. None of the treatments induced locomotor effects in the OFT. These results suggest that DNMTi potentiate the behavioural effects of antidepressant drugs in the FST and that antidepressants, as well as DNMTi, are able to modulate stress-induced changes in DNA methylation in brain regions closely associated with the neurobiology of depression.

  9. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    PubMed

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  10. X-linked Ectodermal Dysplasia Receptor (XEDAR) is Down-regulated in Breast Cancer via Promoter Methylation

    PubMed Central

    Punj, Vasu; Matta, Hittu; Chaudhary, Preet M.

    2009-01-01

    Purpose The X-linked ectodermal dysplasia receptor (XEDAR) is a novel receptor of the Tumor Necrosis Factor Receptor Family that binds to ectodysplasin-A2 (EDA-A2) and induces cell death. The purpose of this study was to determine the tumor-suppressive potential of XEDAR in the development of breast cancer. Experimental Design We analyzed the expression of XEDAR in breast cancer cell lines and tumor samples using quantitative real-time RT-PCR analysis and immunoblotting. We analyzed the human XEDAR gene promoter for the presence of any CpG island and examined its methylation status using methylation-specific real-time PCR. We examined the effect of 5-aza-2′-deoxycytidine (5-Aza-dC) on the expression of XEDAR and sensitivity to EDA-A2-induced apoptosis in breast cancer cell lines. Results Expression of XEDAR, but not EDA-A2, was downregulated in most tumorigenic breast cancer cell lines and tumor samples. Loss of XEDAR expression correlated with the hypermethylation of its promoter. Ectopic expression of XEDAR in MDA-MB-231 cells resulted in significant induction of apoptosis and reduction in colony formation. Treatment with 5-Aza-dC restored XEDAR expression in breast cancer cell lines with methylated XEDAR promoter and sensitized them to EDA-A2-induced cell death. Conclusions Our results suggest that XEDAR expression is down-regulated in most breast cancers via promoter methylation, which may contribute to accelerated tumor development by blocking EDA-A2-induced cell death. XEDAR may represent a novel breast tumor suppressor gene and restoration of its expression by treatment with DNA demethylating agents may represent an attractive approach for the treatment of breast cancer. PMID:20145163

  11. PAQR3 overexpression suppresses the aggressive phenotype of esophageal squamous cell carcinoma cells via inhibition of ERK signaling.

    PubMed

    Bai, Ge; Chu, Jianhu; Eli, Mayinur; Bao, Yongxing; Wen, Hao

    2017-10-01

    Progestin and adipoQ receptor family member 3 (PAQR3) has exhibited anticancer activity in multiple malignancies. However, its expression and function in esophageal squamous cell carcinoma (ESCC) is still elusive. In this work, we examined the expression of PAQR3 in 40 surgically resected ESCC specimens and their adjacent normal tissues. The expression of PAQR3 in ESCC cell lines was measured after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR). The effects of overexpression of PAQR3 on cell proliferation, colony formation, invasion, and tumorigenesis were investigated. It was found that the PAQR3 mRNA level was significantly lower in ESCC than that in adjacent normal tissues (P=0.0318). Low PAQR3 expression was significantly associated with more advanced TNM stage (P=0.0093) and absent lymph node involvement (P=0.0324). Compared to normal esophageal epithelial cells, ESCC cells had significantly lower levels of PAQR3. 5-Aza-CdR treatment led to an induction of PAQR3 in ESCC cells. Enforced expression of PAQR3 significantly inhibited ESCC cell proliferation, colony formation and invasion. Moreover, PAQR3 overexpression blocked cell cycle transition from G1 to S phase, which was associated with induction of p27 and p21 and reduction of cyclin D1, CDK4, and CDK2. Mechanistically, overexpression of PAQR3 suppressed the phosphorylation of ERK1/2 in ESCC cells. In vivo tumorigenic studies confirmed that PAQR3 overexpression retarded the growth of ECA-109 xenograft tumors and inhibited the activation of ERK signaling. Taken together, PAQR3 is epigenetically silenced in ESCC and restoration of PAQR3 suppresses the aggressive phenotype of ESCC cells. Therefore, PAQR3 may represent a potential target for the treatment of ESCC. Copyright © 2017. Published by Elsevier Masson SAS.

  12. Roles of Cell Division and Gene Transcription in the Methylation of CpG Islands

    PubMed Central

    Bender, Christina M.; Gonzalgo, Mark L.; Gonzales, Felicidad A.; Nguyen, Carvell T.; Robertson, Keith D.; Jones, Peter A.

    1999-01-01

    De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methylation of CpG islands located downstream of promoters does not block transcription. We investigated the kinetics of mRNA induction, demethylation, and remethylation of the p16 promoter and second-exon CpG islands in T24 cells after 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment to explore the relationship between CpG island methylation and gene transcription. The rates of remethylation of both CpG islands were associated with time but not with the rate of cell division, and remethylation of the p16 exon 2 CpG island occurred at a higher rate than that of the p16 promoter. We also examined the relationship between the remethylation of coding sequence CpG islands and gene transcription. The kinetics of remethylation of the p16 exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 loci were examined following 5-Aza-CdR treatment because these genes contain exonic CpG islands which are hypermethylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These regions also exhibited higher levels of remethylation in single-cell clones and subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest that de novo methylation is not restricted to the S phase of the cell cycle and that transcription through CpG islands does not inhibit their remethylation. PMID:10490608

  13. Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

    PubMed Central

    Luo, Wei; Karpf, Adam R.; Deeb, Kristin K.; Muindi, Josephia R.; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.

    2010-01-01

    Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. PMID:20587525

  14. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators.

    PubMed

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K

    2012-08-15

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16(INK4a) and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells.

    PubMed

    Tsujioka, Takayuki; Yokoi, Akira; Itano, Yoshitaro; Takahashi, Kentaro; Ouchida, Mamoru; Okamoto, Shuichiro; Kondo, Toshinori; Suemori, Shin-ichiro; Tohyama, Yumi; Tohyama, Kaoru

    2015-11-18

    DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

  16. Germline and somatic DNA methylation and epigenetic regulation of KILLIN in renal cell carcinoma

    PubMed Central

    Bennett, Kristi L.; Campbell, Rebecca; Ganapathi, Shireen; Zhou, Ming; Rini, Brian; Ganapathi, Ram; Neumann, Hartmut P.H.; Eng, Charis

    2011-01-01

    We recently identified germline methylation of KILLIN, a novel p53-regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome-like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis (COBRA) in at least one of the four CpG-rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P<0.0001). Of the 23, 11 (48%) demonstrated methylation in the -598bp to -890bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs 3/23 with germline methylation, p<0.0001). qRT-PCR revealed that methylation significantly downregulates KILLIN expression (P=0.05), and demethylation treatment by 5-aza-2deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic ccRCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents. PMID:21584899

  17. Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

    PubMed Central

    Vorrink, Sabine U.; Hudachek, Danielle R.; Domann, Frederick E.

    2014-01-01

    Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2'-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression. PMID:25116688

  18. A potential role for immunotherapy in thyroid cancer by enhancing NY-ESO-1 cancer antigen expression.

    PubMed

    Gunda, Viswanath; Frederick, Dennie T; Bernasconi, Maria J; Wargo, Jennifer A; Parangi, Sareh

    2014-08-01

    NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells. We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2'-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot. HLA-A2+, NY-ESO-1+ thyroid cell lines were then co-cultured with peripheral blood lymphocytes transduced with NY-ESO-1 specific T-cell receptor (TCR) and assayed for interferon-gamma and Granzyme-B release in the medium. SCID mice injected orthotopically with BCPAP cells were treated with DAC to evaluate for NY-ESO-1 gene expression in vivo. None of the thyroid cancer cell lines showed baseline expression of NY-ESO-1. Three cell lines, BCPAP, TPC-1, and 8505c, showed an increase in NY-ESO-1 gene expression with DAC treatment and were found to be HLA-A2 positive. DAC-treated target BCPAP and TPC-1 tumor cells with up-regulated NY-ESO-1 levels were able to mount an appropriate interferon-gamma and Granzyme-B response upon co-culture with the NY-ESO-1-TCR-transduced peripheral blood lymphocytes. In vivo DAC treatment was able to increase NY-ESO-1 expression in an orthotopic mouse model with BCPAP cells. Our data suggest that many differentiated thyroid cancer cells can be pressed to express immune antigens, which can then be utilized in TCR-based immunotherapeutic interventions.

  19. A Potential Role for Immunotherapy in Thyroid Cancer by Enhancing NY-ESO-1 Cancer Antigen Expression

    PubMed Central

    Gunda, Viswanath; Frederick, Dennie T.; Bernasconi, Maria J.; Wargo, Jennifer A.

    2014-01-01

    Background: NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells. Methods: We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2′-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot. HLA-A2+, NY-ESO-1+ thyroid cell lines were then co-cultured with peripheral blood lymphocytes transduced with NY-ESO-1 specific T-cell receptor (TCR) and assayed for interferon-gamma and Granzyme-B release in the medium. SCID mice injected orthotopically with BCPAP cells were treated with DAC to evaluate for NY-ESO-1 gene expression in vivo. Results: None of the thyroid cancer cell lines showed baseline expression of NY-ESO-1. Three cell lines, BCPAP, TPC-1, and 8505c, showed an increase in NY-ESO-1 gene expression with DAC treatment and were found to be HLA-A2 positive. DAC-treated target BCPAP and TPC-1 tumor cells with up-regulated NY-ESO-1 levels were able to mount an appropriate interferon-gamma and Granzyme-B response upon co-culture with the NY-ESO-1-TCR-transduced peripheral blood lymphocytes. In vivo DAC treatment was able to increase NY-ESO-1 expression in an orthotopic mouse model with BCPAP cells. Conclusion: Our data suggest that many differentiated thyroid cancer cells can be pressed to express immune antigens, which can then be utilized in TCR-based immunotherapeutic interventions. PMID:24811699

  20. Histone Modifications Depict an Aberrantly Heterochromatinized FMR1 Gene in Fragile X Syndrome

    PubMed Central

    Coffee, Bradford; Zhang, Fuping; Ceman, Stephanie; Warren, Stephen T.; Reines, Daniel

    2002-01-01

    Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation but not with chromatin condensation. We also identify alterations in patient cells of two recently identified histone H3 modifications: methylation of histone H3 at lysine 4 and methylation of histone H3 at lysine 9, which are marks for euchromatin and heterochromatin, respectively. In fragile X cells, there is a decrease in methylation of histone H3 at lysine 4 with a large increase in methylation at lysine 9, a change that is consistent with the model of FMR1's switch from euchromatin to heterochromatin in the disease state. The high level of histone H3 methylation at lysine 9 may account for the failure of H3 to be acetylated after treatment of fragile X cells with inhibitors of histone deacetylases, a treatment that fully restores acetylation to histone H4. Using 5-aza-2′-deoxycytidine, we show that DNA methylation is tightly coupled to the histone modifications associated with euchromatin but not to the heterochromatic mark of methylation of histone H3 at lysine 9, consistent with recent findings that this histone modification may direct DNA methylation. Despite the drug-induced accumulation of mRNA in patient cells to 35% of the wild-type level, FMR1 protein remained undetectable. The identification of intermediates in the heterochromatinization of FMR1 has enabled us to begin to dissect the epigenetics of silencing of a disease-related gene in its natural chromosomal context. PMID:12232854

  1. Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation.

    PubMed

    Weisenberger, Daniel J; Velicescu, Mihaela; Cheng, Jonathan C; Gonzales, Felicidad A; Liang, Gangning; Jones, Peter A

    2004-01-01

    Several alternatively spliced variants of DNA methyltransferase (DNMT) 3b have been described. Here, we identified new murine Dnmt3b mRNA isoforms and found that mouse embryonic stem (ES) cells expressed only Dnmt3b transcripts that contained exons 10 and 11, whereas the Dnmt3b transcripts in somatic cells lacked these exons, suggesting that this region is important for embryonic development. DNMT3b2 and 3b3 were the major isoforms expressed in human cell lines and the mRNA levels of these isoforms closely correlated with their protein levels. Although DNMT3b3 may be catalytically inactive, it still may be biologically important because D4Z4 and satellites 2 and 3 repeat sequences, all known DNMT3b target sequences, were methylated in cells that predominantly expressed DNMT3b3. Treatment of cells with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) caused a complete depletion of DNMT1, 3a, 3b1, and 3b2 proteins. Human DNMT3b3 and the murine Dnmt3b3-like isoform, Dnmt3b6, were also depleted although less efficiently, suggesting that DNMT3b3 also may be capable of DNA binding. Moreover, de novo methylation of D4Z4 in T24 cancer cells after 5-Aza-CdR treatment only occurred when DNMT3b3 was expressed, reinforcing its role as a contributing factor of DNA methylation. The expression of either DNMT3b2 or 3b3, however, was not sufficient to explain the abnormal methylation of DNMT3b target sequences in human cancers, which may therefore be dependent on factors that affect DNMT3b targeting. Methylation analyses of immunodeficiency, chromosomal instabilities, and facial abnormalities cells revealed that an Alu repeat sequence was highly methylated, suggesting that Alu sequences are not DNMT3b targets.

  2. Silencing of PCDH10 in hepatocellular carcinoma via de novo DNA methylation independent of HBV infection or HBX expression.

    PubMed

    Fang, Song; Huang, Shi-feng; Cao, Ju; Wen, Yang-an; Zhang, Li-Ping; Ren, Guo-Sheng

    2013-05-01

    PCDH10 is a key tumor suppressive gene for nasopharyngeal, esophageal, and other carcinomas with frequent methylation. In this study, we investigated the potential epigenetic modification of the PCDH10 gene by hepatitis B virus × protein (HBx), a pivotal factor in the progression of HBV replication and potential carcinogenesis. PCDH10 expression was found to be down-regulated in 9/13 (69.2 %) of hepatocellular carcinoma (HCC) cell lines. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) was sufficient to restore PCDH10 mRNA expression by suppressing PCDH10 promoter methylation in HepG2 cells. Treatment with Trichostatin A alone had no significant effect on PCDH10 expression but enhanced the effect of Aza. PCDH10 methylation was further detected in 76 % (38 of 50) of HCC tissues compared with 40 % (20 of 50) of paired adjacent tissues, with no methylation detected in normal human liver tissues. There were significant correlations between methylation status of PCDH10 and tumor size, serum AFP levels, metastasis or TNM staging (P < 0.05). Moreover, PCDH10 promoter methylation status was not associated with HBV infection in our panel of 50 primary HCC tumors, and transfection with HBX could not alter the status of PCDH10 promoter methylation. Collectively, these observations suggested that the expression of PCDH10 was silenced in HCC via de novo DNA methylation independent of HBV infection or HBX expression, and PCDH10 might form a potentially useful therapeutic target for HCC.

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

    PubMed

    Perazzoli, Gloria; Prados, Jose; Ortiz, Raul; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    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. 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. 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. 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 multiforme patients.

  4. A New Class of Quinoline-Based DNA Hypomethylating Agents Reactivates Tumor Suppressor Genes by Blocking DNA Methyltransferase 1 Activity and Inducing Its Degradation

    PubMed Central

    Datta, Jharna; Ghoshal, Kalpana; Denny, William A.; Gamage, Swarna A.; Brooke, Darby G.; Phiasivongsa, Pasit; Redkar, Sanjeev; Jacob, Samson T.

    2010-01-01

    Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2′-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC50 (6–13 µ mol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 µmol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027–induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy. PMID:19417133

  5. Rescued expression of WIF-1 in gallbladder cancer inhibits tumor growth and induces tumor cell apoptosis with altered expression of proteins

    PubMed Central

    Huang, Yan; Du, Qiang; Wu, Weibao; She, Feifei; Chen, Yanling

    2016-01-01

    As a highly conserved metabolic pathway, the Wnt signaling pathway is involved in cell differentiation, proliferation and several other processes. In normal cells, this pathway is suppressed, and abnormal activation is often associated with tumor occurrence and development. In certain types of tumor, Wnt inhibitory factor 1 (WIF-1), an inhibitor of the Wnt pathway, inhibits tumor growth. However, the effect of the expression of WIF-1 on gallbladder cancer remains to be fully elucidated. In the current study, reverse transcription-quantitative polymerase chain reaction and western blotting were conducted. The present study demonstrated that, in gallbladder cancer, WIF-1 generally exhibited low levels of expression as a result of gene promoter methylation. Treatment with the drug, 5-aza-2-deoxycytidine, increased the expression of WIF-1 in the GBC-SD gallbladder cell line. In addition, a WIF-1-expression plasmid was transfected into GBC-SD cells, and it was found that cell proliferation, invasion and metastasis declined significantly, whereas the apoptotic rate increased. A nude mouse tumor transplantation experiment showed that the oncogenicity of the GBC-SD cells expressing WIF-1 was substantially lower, compared with that of the untransfected GBC-SD cells and of GBD-SD cells expressing the control plasmid. A fluorescent protein chip experiment showed that the restored expression of WIF-1 affected the expression of several cellular proteins. These alterations may explain the different biological behavior of the tumor cells expressing WIF-1. As an effective inhibitory factor of the Wnt signaling pathway, WIF-1 modulated the expression of proteins controlling the proliferation, apoptosis and metastasis of gallbladder tumor cells, thus suppressing the tumor. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer. PMID:27430608

  6. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice.

    PubMed

    Terse, Pramod; Engelke, Kory; Chan, Kenneth; Ling, Yonghua; Sharpnack, Douglas; Saunthararajah, Yogen; Covey, Joseph M

    2014-01-01

    Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity

  7. Methylated Bone Morphogenetic Protein 3 (BMP3) Gene: Evaluation of Tumor Suppressor Function and Biomarker Potential in Biliary Cancer

    PubMed Central

    Kisiel, John B; Li, Jia; Zou, Hongzhi; Oseini, Abdul M; Strauss, Benjamin B; Gulaid, Kadra H.; Moser, Catherine D; Aderca, Ileana; Ahlquist, David A; Roberts, Lewis R; Shire, Abdirashid M

    2014-01-01

    Background Although cholangiocarcinoma (CC) is an uncommon and highly lethal malignancy, early detection enables the application of potentially curative therapies and improves survival. Consequently, tools to improve the early diagnosis of CC are urgently needed. During a screen for genes epigenetically suppressed by methylation in CC that might serve as methylation markers for CC, we found that the BMP3 gene is methylated in CC cell lines, but the potential diagnostic value and the function of BMP3 in CC are unknown. Methods We aimed to quantitatively assess BMP3 methylation in resected CC tumor specimens using methylation specific PCR and evaluate the tumor suppressor role of BMP3 in biliary cancer cell lines in comparison to an immortalized normal cholangiocyte cell line. Expression of BMP3 was quantified by mRNA levels before and after treatment with 5-Aza-2’-deoxycytidine and trichostatin A. After transfection with a BMP3-containing plasmid, cell viability was measured using the bromodeoxyuridine incorporation assay and apoptosis quantified by caspase assay. Results In primary CC tumor tissue specimens significantly more methylated BMP3 copies were found when compared to matched benign bile duct epithelium from the same patient, with high specificity. BMP3 expression was absent in cell lines with BMP3 methylation; this suppression of BMP3 expression was reversed by treatment with a DNA demethylating agent and histone de-acetylase inhibitor. Transfection of a BMP3-expressing construct into a BMP3-negative biliary cancer cell line restored BMP3 mRNA expression and reduced cell proliferation and cell viability while increasing the rate of apoptosis. Conclusion These findings strongly support a tumor suppressor role for BMP3 in CC and suggest that BMP3 methylation may be a new biomarker for early detection of CCs. of the peptidome are also involved. PMID:25077038

  8. Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent stem cells

    PubMed Central

    Wesemann, Duane R.; Portuguese, Andrew J.; Magee, Jennifer M.; Gallagher, Michael P.; Zhou, Xiaolong; Panchakshari, Rohit A.; Alt, Frederick W.

    2012-01-01

    Induced pluripotent stem cells (iPSCs) can be formed from somatic cells by a defined set of genetic factors; however, aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster often hinders their developmental potency and ability to contribute to high-grade chimerism in mice. Here, we describe an approach that allows splenic B cells activated to undergo Ig heavy-chain (IgH) class-switch recombination (CSR) to be reprogrammed into iPSCs that contribute to high-grade chimerism in mice. Treatment of naïve splenic B cells in culture with anti-CD40 plus IL-4 induces IgH CSR from IgM to IgG1 and IgE. CSR leads to irreversible IgH locus deletions wherein the IgM-producing Cμ exons are permanently excised from the B-cell genome. We find that anti-CD40 plus IL-4–activated B cells produce iPSCs that are uniformly hypermethylated in the imprinted Dlk1-Dio3 gene cluster and fail to produce chimerism in mice. However, treatment of activated B cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine before and at early stages of reprogramming attenuates hypermethylation of the Dlk1-Dio3 locus in resultant iPSCs and enables them to form high-grade chimerism in mice. These conditions allowed us to produce chimeric mice in which all mature B cells were derived entirely from IgG1-expressing B-cell–derived iPSCs. We conclude that culture conditions of activated B cells before and at early stages of reprogramming influence the developmental potency of resultant iPSCs. PMID:22869756

  9. Enhanced memory persistence is blocked by a DNA methyltransferase inhibitor in the snail Lymnaea stagnalis.

    PubMed

    Lukowiak, Ken; Heckler, Benjamin; Bennett, Thomas E; Schriner, Ellen K; Wyrick, Kathryn; Jewett, Cynthia; Todd, Ryan P; Sorg, Barbara A

    2014-08-15

    Lymnaea stagnalis provides an excellent model system for studying memory because these snails have a well-described set of neurons, a single one of which controls expression of long-term memory of operantly conditioned respiratory behavior. We have shown that several different manipulations, including pre-training exposure to serotonin (5-HT) or methamphetamine, submersion of snails after training to prevent memory interference, and exposure to effluent from predatory crayfish (CE), enhance memory persistence. Changes in DNA methylation underlie formation of strong memories in mammals and 5-HT-enhanced long-term facilitation in Aplysia. Here we determined the impact of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-AZA; 87 μmol l(-1)), on enhanced memory persistence by all four manipulations. We found that 5-HT (100 μmol l(-1)) enhanced memory persistence, which was blocked by 5-AZA pretreatment. Snails pre-exposed to 3.3 μmol l(-1) Meth 4 h prior to training demonstrated memory 72 h later, which was not present in controls. This memory-enhancing effect was blocked by pre-treatment with 87 μmol l(-1) 5-AZA. Similarly, submersion to prevent interference learning as well as training in CE produced memory that was not present in controls, and these effects were blocked by pre-treatment with 87 μmol l(-1) 5-AZA. In contrast, 5-AZA injection did not alter expression of normal (non-enhanced) memory, suggesting that these four stimuli enhance memory persistence by increasing DNA methyltransferase activity, which, in turn, increases expression of memory-enhancing genes and/or inhibits memory suppressor genes. These studies lay important groundwork for delineating gene methylation changes that are common to persistent memory produced by different stimuli.

  10. Epigenetic silencing of BTB and CNC homology 2 and concerted promoter CpG methylation in gastric cancer.

    PubMed

    Haam, Keeok; Kim, Hee-Jin; Lee, Kyung-Tae; Kim, Jeong-Hwan; Kim, Mirang; Kim, Seon-Young; Noh, Seung-Moo; Song, Kyu-Sang; Kim, Yong Sung

    2014-09-01

    BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription factor with a prominent role in B-cell development. Genetic polymorphisms within a single locus encoding BACH2 are associated with various autoimmune diseases and allergies. In this study, restriction landmark genomic scanning revealed methylation at a NotI site in a CpG island covering the BACH2 promoter in gastric cancer cell lines and primary gastric tumors. Increased methylation of the BACH2 promoter was observed in 52% (43/83) of primary gastric tumors, and BACH2 hypermethylation was significantly associated with decreased gene expression. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin. A restored BACH2 expression in BACH2-silenced gastric cancer cell lines, and knockdown of BACH2 using short hairpin RNA (i.e. RNA interference) increased cell proliferation in gastric cancer cells. Clinicopathologic data showed that decreased BACH2 expression occurred significantly more frequently in intestinal-type (27/44, 61%) compared with diffuse-type (13/50, 26%) gastric cancers (P<0.001). Furthermore, BACH2 promoter methylation paralleled that of previously identified targets, such as LRRC3B, LIMS2, PRKD1 and POPDC3, in a given set of gastric tumors. We propose that concerted methylation in many promoters plays a role in accelerating gastric tumor formation and that methylated promoter loci may be targets for therapeutic treatment, such as the recently introduced technique of epigenetic editing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. Absence of Metallothionein 3 Expression in Breast Cancer is a Rare, But Favorable Marker of Outcome that is Under Epigenetic Control

    PubMed Central

    Somji, Seema; Garrett, Scott H.; Zhou, Xu Dong; Zheng, Yun; Sens, Donald A.; Sens, Mary Ann

    2010-01-01

    Cadmium (Cd+2), a known carcinogen mimics the effects of estrogen in the uterus and mammary gland suggesting its possible involvement in the development and progression of breast cancer. This lab showed through analysis of a small set of archival human diagnostic specimens that the third isoform of the classic Cd+2 binding protein metallothionein (MT-3), is not expressed in normal breast tissue, but is expressed in some breast cancers and that expression tends to correlate with a poor disease outcome. The goals of the present study were to verify that overexpression of MT-3 in a large set of archival human diagnostic specimens tends to correlate with poor disease outcome and define the mechanism of MT-3 gene regulation in the normal breast epithelial cell. The results showed that MT-3 was expressed in approximately 90% of all breast cancers and was absent in normal breast epithelium. The lack of MT-3 staining in some cancers correlated with a favorable patient outcome. High frequency of MT-3 staining was also found for in situ breast cancer suggesting that MT-3 might be an early biomarker for breast cancer. The study also demonstrated that the MCF-10A cell line, an immortalized, non-tumorigenic model of human breast epithelial cells, displayed no basal expression of MT-3, nor was it induced by Cd+2. Treatment of the MCF-10A cells with the demethylation agent, 5-Aza-2′-deoxycytidine, or the histone deacetylase inhibitor, MS-275, restored MT-3 mRNA expression. It was also shown that the MT-3 metal regulatory elements are potentially active binders of protein factors following treatment with these inhibitors suggesting that MT-3 expression may be subject to epigenetic regulation. PMID:21170156

  12. The intracellular activation of lamivudine (3TC) and determination of 2′-deoxycytidine-5′-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells

    PubMed Central

    Kewn, Stephen; Hoggard, Patrick G; Sales, Sean D; Johnson, Mark A; Back, David J

    2000-01-01

    Aims Lamivudine (3TC, 2′-deoxy-3′-thiacytidine) requires intracellular metabolism to its active 5′-triphosphate, 3TC-5′-triphosphate (3TCTP), to inhibit the replication of hepatitis B virus (HBV). We have investigated the activation of 3TC, in the presence and absence of a range of compounds, in HepG2 cells. The intracellular levels of the endogenous competitor of 3TCTP, 2′-deoxycytidine-5′-triphosphate (dCTP), were also determined and 3TCTP/dCTP ratios calculated. Methods The effects of a number of compounds on 3TC (3H; 1 μM) phosphorylation were investigated by radiometric h.p.l.c. dCTP levels were determined using a template primer extension assay. 3TCTP/dCTP ratios were calculated from these results. Results The phosphorylation of 3TC was significantly increased in the presence of either hydroxyurea (HU), methotrexate (MTX), or fludarabine (FLU). For example, at 100 μm HU, control 3TCTP levels were increased to 361% of control, whereas at 100 μm FLU, control 3TCTP levels were increased to 155%. dCTP pools were significantly reduced in the presence of HU and FLU, at 100 μm concentrations only. However, for all the above three compounds investigated, the ratio of 3TCTP/dCTP was favourably enhanced (e.g. at 1 μm MTX, 255% of control). Neither ganciclovir (GCV), lobucavir (LCV), penciclovir (PCV), adefovir dipivoxil (ADV), nor foscarnet (FOS) had any significant effects on 3TC phosphorylation or dCTP pools. Conclusions These results suggest that the activity of 3TC may be potentiated when combined with one of the modulators studied. The lack of an interaction between 3TC and the other anti-HBV agents is reassuring. These in vitro studies can be used as an initial screen to examine potential interactions at the phosphorylation level. PMID:11136299

  13. Reduced expression of GNA11 and silencing of MCT1 in human breast cancers.

    PubMed

    Asada, Kiyoshi; Miyamoto, Kazuaki; Fukutomi, Takashi; Tsuda, Hitoshi; Yagi, Yukiko; Wakazono, Kuniko; Oishi, Shoko; Fukui, Hiroshi; Sugimura, Takashi; Ushijima, Toshikazu

    2003-01-01

    Alteration in the methylation status of a gene is often associated with its altered expression. Based on a genome scanning technique for differences in CpG methylations, methylation-sensitive representational difference analysis, DNA fragments hypermethylated in a human breast cancer were isolated. A DNA fragment was isolated from intron 1 of guanine-nucleotide-binding protein alpha-11 (GNA11). mRNA expression of GNA11 was shown to be decreased in 10 of 16 breast cancers by RT-PCR analysis, and the immunoreactivity of the GNA11 product, Galpha11 subunit of heterotrimeric G-protein, was observed to be reduced in 14 of the 16 cancers by immunohistochemistry. Methylation of a CpG island (CGI) in the 5' region of GNA11 or that of intron 1 did not show a clear correlation with its decreased expression. Another DNA fragment was isolated from a CGI in the 5' upstream region of monocarboxylate transporter 1 (MCT1), and was methylated in 4 of 20 breast cancers. The CGI was also methylated in a human breast cancer cell line, MDA-MB-231, and quantitative RT-PCR showed that its expression was almost lost in the cell line. By treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine, the methylation was removed and the expression was restored. GNA11 is involved in signalling of gonadotropin-releasing hormone receptor, which negatively regulates cell growth. MCT1 is involved in cellular transportation of butyrate, which induces cellular differentiation. Downregulation of these two genes was suggested to be involved in human breast cancers. Copyright 2003 S. Karger AG, Basel

  14. hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

    PubMed Central

    Anwar, Sumadi Lukman; Krech, Till; Hasemeier, Britta; Schipper, Elisa; Schweitzer, Nora; Vogel, Arndt; Kreipe, Hans; Buurman, Reena; Skawran, Britta; Lehmann, Ulrich

    2017-01-01

    AIM To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC). METHODS Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables. RESULTS miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes. CONCLUSION Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker. PMID:28321157

  15. The Human ARF Cell Cycle Regulatory Gene Promoter Is a CpG Island Which Can Be Silenced by DNA Methylation and Down-Regulated by Wild-Type p53

    PubMed Central

    Robertson, Keith D.; Jones, Peter A.

    1998-01-01

    The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check. PMID:9774662

  16. Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

    PubMed

    Kitazawa, Riko; Kitazawa, Sohei

    2007-01-01

    Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

  17. Identification of novel tumor markers in prostate, colon and breast cancer by unbiased methylation profiling.

    PubMed

    Chung, Woonbok; Kwabi-Addo, Bernard; Ittmann, Michael; Jelinek, Jaroslav; Shen, Lanlan; Yu, Yinhua; Issa, Jean-Pierre J

    2008-04-30

    DNA hypermethylation is a common epigenetic abnormality in cancer and may serve as a useful marker to clone cancer-related genes as well as a marker of clinical disease activity. To identify CpG islands methylated in prostate cancer, we used methylated CpG island amplification (MCA) coupled with representational difference analysis (RDA) on prostate cancer cell lines. We isolated 34 clones that corresponded to promoter CpG islands, including 5 reported targets of hypermethylation in cancer. We confirmed the data for 17 CpG islands by COBRA and/or pyrosequencing. All 17 genes were methylated in at least 2 cell lines of a 21-cancer cell line panel containing prostate cancer, colon cancer, leukemia, and breast cancer. Based on methylation in primary tumors compared to normal adjacent tissues, NKX2-5, CLSTN1, SPOCK2, SLC16A12, DPYS and NSE1 are candidate biomarkers for prostate cancer (methylation range 50%-85%). The combination of NSE1 or SPOCK2 hypermethylation showed a sensitivity of 80% and specificity of 95% in differentiating cancer from normal. Similarly NKX2-5, SPOCK2, SLC16A12, DPYS and GALR2 are candidate biomarkers for colon cancer (methylation range 60%-95%) and GALR2 hypermethylation showed a sensitivity of 85% and specificity of 95%. Finally, SLC16A12, GALR2, TOX, SPOCK2, EGFR5 and DPYS are candidate biomarkers for breast cancer (methylation range 33%-79%) with the combination of EGFR5 or TOX hypermethylation showing a sensitivity of 92% and specificity of 92%. Expression analysis for eight genes that had the most hypermethylation confirmed the methylation associated silencing and reactivation with 5-aza-2'-deoxycytidine treatment. Our data identify new targets of transcriptional silencing in cancer, and provide new biomarkers that could be useful in screening for prostate cancer and other cancers.

  18. MARVELD1 inhibited cell proliferation and enhance chemosensitivity via increasing expression of p53 and p16 in hepatocellular carcinoma.

    PubMed

    Yu, Youtao; Zhang, Yubao; Hu, Jianran; Zhang, Hao; Wang, Shan; Han, Fang; Yue, Lei; Qu, Youpeng; Zhang, Yao; Liang, Hongjian; Nie, Huan; Li, Yu

    2012-04-01

    We have previously found that expression of MARVELD1 was remarkably downregulated in multiple tumor tissues, but unclear in hepatocellular carcinoma (HCC) and its function has not been explored yet. In the present study, to uncover the underlying mechanism of MARVELD1 in the pathogenesis and development of HCC, we investigated the expression pattern of MARVELD1 and its effect on tumor proliferation in HCC. The results indicated the frequent downregulation of MARVELD1 in clinic samples and cell lines of HCC resulted from promoter methylation, as well as genetic deletion. Furthermore, treatment of MARVELD1 unexpressing Hep3B2.1-7 and PLC/PRF/5 cells with the demethylating agent 5-aza-2' deoxycytidine restored its expression. Overexpression of MARVELD1 suppressed the proliferation of HCC cells in vitro and in vivo, whereas downregulation of endogenous MARVELD1 by shRNAs significantly enhanced these characters. MARVELD1 overexpression could enhance chemosensitivity of HCC cells to epirubicin and 10-hydroxycamptothecin. Corresponding to these results, the expression of p-ERK1/2 and cyclin D1 were decreased, whereas p16 and p53 were increased in MARVELD1-transfected cells. We also demonstrated that knockdown of MARVELD1 resulted in upregulation of p-ERK1/2 and cyclin D1, and downregulation of p16 and p53. Moreover, the effect of the decreased cell growth rate was significantly reversed when MARVELD1-overexpressing cells were trasfected with p53 or p16 siRNA. Our findings suggest that MARVELD1 is a tumor suppressor by negatively regulating proliferation, tumor growth and chemosensitivity of HCC cells via increasing p53 and p16 in vitro and in vivo. MARVELD1 may be a potential target for HCC therapy. © 2012 Japanese Cancer Association.

  19. Epigenetic inactivation of protein kinase D1 in gastric cancer and its role in gastric cancer cell migration and invasion.

    PubMed

    Kim, Mirang; Jang, Hay-Ran; Kim, Jeong-Hwan; Noh, Seung-Moo; Song, Kyu-Sang; Cho, June-Sik; Jeong, Hyun-Yong; Norman, Jim C; Caswell, Patrick T; Kang, Gyeong Hoon; Kim, Seon-Young; Yoo, Hyang-Sook; Kim, Yong Sung

    2008-03-01

    Protein kinase D (PKD) 1 influences cell migration by mediating both trans-Golgi vesicle fission and integrin recycling to the cell surface. Using restriction landmark genomic scanning methods, we found that the promoter region of PKD1 was aberrantly methylated in gastric cancer cell lines. Silencing of PKD1 expression was detected in 72.7% of gastric cancer cell lines examined, and the silencing was associated with CpG hypermethylation in the promoter region of PKD1. Treatment with 5-aza-2'-deoxycytidine and trichostatin A partially reversed PKD1 methylation and restored gene expression in PKD1-silenced cell lines. Real-time reverse transcription-polymerase chain reaction analysis of 96 paired clinical primary gastric cancer samples revealed that 59% of the analyzed tumors had a >2-fold decrease in PKD1 expression compared with each normal-appearing tissue and that this downregulation of PKD1 expression was significantly correlated with increased methylation. We also observed a gradual increase in the level of promoter methylation of PKD1 in aging, normal-appearing mucosal tissues, suggesting that PKD1 methylation may be one of the earliest events that predispose an individual to gastric cancer. PKD1 expression was required for directional migration of gastric cancer cells. Furthermore, knock down of PKD1 by RNA interference promoted the invasiveness of cell lines that expressed PKD1 at relatively high levels. Based on these results, we propose that PKD1 is frequently silenced by epigenetic regulation, which plays a role in cell migration and metastasis in gastric cancer.

  20. Epigenetic silencing of the chaperone Cosmc in human leukocytes expressing tn antigen.

    PubMed

    Mi, Rongjuan; Song, Lina; Wang, Yingchun; Ding, Xiaokun; Zeng, Junwei; Lehoux, Sylvain; Aryal, Rajindra P; Wang, Jianmei; Crew, Vanja K; van Die, Irma; Chapman, Arlene B; Cummings, Richard D; Ju, Tongzhong

    2012-11-30

    Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1-3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2'-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer.

  1. Epigenetic Regulation of miR-29s Affects the Lactation Activity of Dairy Cow Mammary Epithelial Cells.

    PubMed

    Bian, Yanjie; Lei, Yu; Wang, Chunmei; Wang, Jie; Wang, Lina; Liu, Lili; Liu, Lixin; Gao, Xuejun; Li, Qingzhang

    2015-09-01

    Milk is important for human nutrition, and enhanced milk quality has become a major selection criterion for the genetic improvement of livestock. Epigenetic modifications have been shown to be involved in mammary gland development; but the mechanisms underlying their effects remain unknown. MicroRNAs are involved in the regulation of milk synthesis and in mammary gland development. Our study is the first to investigate the roles of miR-29s and epigenetic regulation in dairy cow mammary epithelial cells (DCMECs). Our results show that miR-29s regulate the DNA methylation level by inversely targeting both DNMT3A and DNMT3B in DCMECs. The inhibition of miR-29s caused global DNA hypermethylation and increased the methylation levels of the promoters of important lactation-related genes, including casein alpha s1 (CSN1S1), E74-like factor 5 (ElF5), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein-1 (SREBP1), and glucose transporter 1 (GLUT1). The inhibition of miR-29s reduced the secretion of lactoprotein, triglycerides (TG) and lactose by DCMECs. Moreover, the treatment of DCMECs with 5-aza-2'-deoxycytidine (5-Aza-dC) decreased the methylation levels of the miR-29b promoter and increased the expression of miR-29b. The link between miR-29s and DNMT3A/3B enhances our understanding of the roles of miRNAs in mammary gland function, and our data will inform more experimentally oriented studies to identify new mechanisms of regulating lactation. We present new insights regarding the epigenetic regulation of lactation performance. Improved understanding of the molecular basis of lactation will aid in the development of strategies for optimizing milk quality in dairy cows and modifying the lactation performance of offspring.

  2. Epigenetic Silencing of the Chaperone Cosmc in Human Leukocytes Expressing Tn Antigen*

    PubMed Central

    Mi, Rongjuan; Song, Lina; Wang, Yingchun; Ding, Xiaokun; Zeng, Junwei; Lehoux, Sylvain; Aryal, Rajindra P.; Wang, Jianmei; Crew, Vanja K.; van Die, Irma; Chapman, Arlene B.; Cummings, Richard D.; Ju, Tongzhong

    2012-01-01

    Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1–3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2′-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer. PMID:23035125

  3. Epigenetic Regulation of MicroRNA Genes and the Role of miR-34b in Cell Invasion and Motility in Human Melanoma

    PubMed Central

    Mazar, Joseph; Khaitan, Divya; DeBlasio, Dan; Zhong, Cuncong; Govindarajan, Subramaniam S.; Kopanathi, Sharmila; Zhang, Shaojie; Ray, Animesh; Perera, Ranjan J.

    2011-01-01

    Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2′-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis. PMID:21949788

  4. Epstein-Barr virus down-regulates tumor suppressor DOK1 expression.

    PubMed

    Siouda, Maha; Frecha, Cecilia; Accardi, Rosita; Yue, Jiping; Cuenin, Cyrille; Gruffat, Henri; Manet, Evelyne; Herceg, Zdenko; Sylla, Bakary S; Tommasino, Massimo

    2014-05-01

    The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2'-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.

  5. Epstein-Barr Virus Down-Regulates Tumor Suppressor DOK1 Expression

    PubMed Central

    Siouda, Maha; Frecha, Cecilia; Accardi, Rosita; Yue, Jiping; Cuenin, Cyrille; Gruffat, Henri; Manet, Evelyne; Herceg, Zdenko; Sylla, Bakary S.; Tommasino, Massimo

    2014-01-01

    The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2′-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis. PMID:24809689

  6. ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma

    PubMed Central

    2009-01-01

    Background Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes. Methods In a methylation screening approach, we identified ECRG4 as a differentially methylated gene. We analyzed different cancer cells for ECRG4 promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The ECRG4 coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an ECRG4-eGFP fusion gene. Results We found a high frequency of ECRG4 promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of ECRG4 was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. ECRG4 hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated in vitro by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of ECRG4 in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium. Conclusions ECRG4 is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule. PMID:20017917

  7. The long non-coding RNA maternally expressed gene 3 activates p53 and is downregulated in esophageal squamous cell cancer.

    PubMed

    Lv, Desheng; Sun, Run; Yu, Qian; Zhang, Xuefei

    2016-10-24

    Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor survival. Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression; hence, lncRNAs are also involved in the development and progression of ESCC. In this study, we used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression of lncRNA, maternally expressed gene 3 (MEG3) in ESCC. Ectopic expression of MEG3 was performed in ESCC cell lines. Proliferation and apoptosis of ESCC cell lines were analyzed after ectopic expression of MEG3. We found MEG3 was significantly downregulated in ESCC tissues compared with normal tissues by qRT-PCR. Low expression of MEG3 was correlated with lymph node metastasis and advanced TNM stages of ESCC patients and indicated shorter survival (HR = 0.471, 95 % CI 0.234-0.950, P = 0.035), which was confirmed by The Cancer Genome Atlas (TCGA) esophageal cancer dataset. DNA-demethylating agent (5-aza-2-deoxy-cytidine (5-aza-CdR)) treatment significantly increased MEG3 expression level in ESCC cells, and TCGA esophageal cancer dataset also showed that DNA methylation of MEG3 predicted survival. Ectopic expression of MEG3 in ESCC cells inhibited cell proliferation, promoted apoptosis, and suppressed metastasis. Further investigation showed enforced expression of MEG3 activated p53 and its target genes by downregulation of mouse double minute 2 homolog (MDM2). Overall, our study indicated that MEG3 expression loss is common in ESCC and MEG3 could activate p53 and predict prognosis in ESCC.

  8. Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells by directly targeting PRL-3.

    PubMed

    Li, Zhengrong; Zhang, Guoyang; Li, Daojiang; Jie, Zhigang; Chen, Heping; Xiong, Jianbo; Liu, Yi; Cao, Yi; Jiang, Mengmeng; Le, Zhibiao; Tan, Shengxing

    2015-01-02

    Phosphatase of regenerating liver-3 (PRL-3) is believed to be associated with cell motility, invasion, and metastasis. Our previous work found that PRL-3 is highly overexpressed in gastric cancer (GC) tissue with peritoneal metastasis and directly involved in the pathogenesis of GC peritoneal metastasis. Moreover, we further found that the down-regulation of endogenous miR-495 expression plays a causative role in over expression of PRL-3 in GC peritoneal metastasis. However, the molecular regulation mechanisms by which endogenous miR-495 expression is down-regulated and PRL-3 promotes GC peritoneal metastasis remain to be clearly elucidated. Some studies have shown that the promoter methylation is closely related to the miRNA gene expression. Therefore, in present study, based on our previous findings, we will analysis whether DNA methylation is a major cause of the down-expression of endogenous miR-495, which results in PRL-3 overexpression in GC peritoneal metastasis. Methylation specific PCR (MSP) and sodium bisulfite sequencing method (BSP) detected miR-495 gene promoter methylation status. We treated GC cell lines with 5-Aza-2'-deoxycytidine (5-Aza-dC) to make the gene promoter methylation inactivation. By treating with 5-Aza-dC the migration and invasion of GC cells were significantly inhibited. And the miR-495 was overexpressing, corresponds to the mRNA and protein levels of PRL-3 were reduced, the ability of invasion and metastasis was inhibited. This study suggest that miR-495 have tumor suppressor properties and are partially silenced by DNA hypermethylation in GC, will provide new strategies for prevention and treatment of GC peritoneal metastasis.

  9. HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer.

    PubMed

    Hwang, Jung-Ah; Lee, Bo Bin; Kim, Yujin; Hong, Seung-Hyun; Kim, Young-Ho; Han, Joungho; Shim, Young Mog; Yoon, Chae-Yeong; Lee, Yeon-Su; Kim, Duk-Hwan

    2015-06-01

    This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (P<0.0001; Wilcoxon signed-rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P=0.12) and Ki-67 proliferation index (P=0.15). However, patients with HOXA9 hypermethylation had poor recurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC.

  10. Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis

    PubMed Central

    Connelly, Jessica J.; Cherepanova, Olga A.; Doss, Jennifer F.; Karaoli, Themistoclis; Lillard, Travis S.; Markunas, Christina A.; Nelson, Sarah; Wang, Tianyuan; Ellis, Peter D.; Langford, Cordelia F.; Haynes, Carol; Seo, David M.; Goldschmidt-Clermont, Pascal J.; Shah, Svati H.; Kraus, William E.; Hauser, Elizabeth R.; Gregory, Simon G.

    2013-01-01

    Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by ‘genomic convergence’ for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2′-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex disease. PMID:23912340

  11. Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

    PubMed Central

    Han, Yoo-Jeong; Zhang, Jing; Zheng, Yonglan; Huo, Dezheng; Olopade, Olufunmilayo I.

    2016-01-01

    Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner. PMID:27806084

  12. Myogenic Differentiation Potential of Mesenchymal Stem Cells Derived from Fetal Bovine Bone Marrow.

    PubMed

    Okamura, Lucas Hidenori; Cordero, Paloma; Palomino, Jaime; Parraguez, Victor Hugo; Torres, Cristian Gabriel; Peralta, Oscar Alejandro

    2017-03-07

    The myogenic potential of bovine fetal MSC (bfMSC) derived from bone marrow (BM) remains unknown; despite its potential application for the study of myogenesis and its implications for livestock production. In the present study, three protocols for in vitro myogenic differentiation of bfMSC based on the use of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza), myoblast-secreted factor Galectin-1 (Gal-1), and myoblast culture medium SkGM-2 BulletKit were used. Plastic-adherent bfMSC were isolated from fetal BM collected from abattoir-derived fetuses. Post-thaw viability analyses detected 85.6% bfMSC negative for propidium iodine (PI). Levels of muscle regulatory factors (MRF) MYF5, MYF6, MYOD, and DES mRNA were higher (P < 0.05) in bfMSC cultured under 100 µM of 5-Aza compared to 1 and 10 µM. Treatment of bfMSC with 10 µM of 5-Aza resulted in down-regulation of MYOD mRNA (Days 7 to 21) and up-regulation of MYF6 (Day 7), MYF5, and DES mRNA (Day 21). Gal-1 and SkGM-2 BulletKit induced sequential down-regulation of early MRF (MYF5) and up-regulation of intermediate (MYOD) and late MRF (DES) mRNA. Moreover, DES and MYF5 were immunodetected in differentiated bfMSC. In conclusion, protocols evaluated in bfMSC induced progress into myogenic differentiation until certain extent evidenced by changes in MRF gene expression.

  13. Methylation pattern of H19 exon 1 is closely related to preeclampsia and trophoblast abnormalities.

    PubMed

    Lu, Linshan; Hou, Zheng; Li, Li; Yang, Yanhong; Wang, Xiaohong; Zhang, Beilei; Ren, Mo; Zhao, Dan; Miao, Zhuo; Yu, Lili; Yao, Yuanqing

    2014-09-01

    Preeclampsia (PE) is a pregnancy-induced disorder characterized by the overproliferation of trophoblasts. Hydatidiform moles, which are associated with a high risk of developing PE, are characterized by the excessive proliferation of trophoblastic tissue. H19 is highly expressed in placental tissue; however, its biological function remains unclear. A fundamental modification of the H19 gene is DNA methylation, which typically occurs in CG-rich regions at the promoter or the first exon region. In this study, in order to investigate the DNA methylation pattern of the H19 exon 1 region in placental tissues and trophoblast cells, placental specimens were collected from women in the first trimester of pregrancy (FTP) and the third trimester of pregnancy (TTP), as well as from from women with severe preeclampsia (sPE). We found that the DNA methylation levels of H19 exon 1 were significantly higher in the tissues obtained from women in TTP than from those obtained from women in FFP. The methylation status of CpG 1 sites within exon 1 of H19 was markedly higher in the placental tissues obtained from women with sPE than in the tissues obtained from women in TTP. In addition, we used the human choriocarcinoma cell line, JEG-3, and treated the cells with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza‑Dc). Following treatment with 5-Aza-Dc, the methylation levels at this CpG site showed marked hypomethylation. In addtion, the cell proliferative, migratory and invasive capacities of the cells were remarkably inhibited. Our data suggest that hypermethylation at individual CpG sites within exon 1 of H19 may be involved in the dysfunction of trophoblasts and the pathogenesis of PE.

  14. The expression of MAGE-C1 and MAGE-C2 in breast cancer and their clinical significance.

    PubMed

    Hou, Shuyun; Sang, Meixiang; Zhao, Lianmei; Hou, Ran; Shan, Baoen

    2016-01-01

    Our study aims to analyze the expression pattern, mechanism, and prognostic significance of melanoma-associated antigen MAGE-C1 and MAGE-C2 in breast cancer. Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to investigate the expressions of MAGE-C1 and MAGE-C2 in breast benign disease specimens, tumor-free breast specimens, and breast cancer specimens; their correlation with clinicopathologic parameters and recurrence-free survival was elucidated. We examined the influence of DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) together with histone deacetylase inhibitor trichostatin A on the expression of MAGE-C1 and MAGE-C2 in breast cancer cell lines. Proteins for MAGE-C1 and MAGE-C2 expressions were 38.3% and 58.3% in breast cancer specimens, messenger RNA for MAGE-C1 and MAGE-C2 expressions were 43.3% and 61.7%, respectively. MAGE-C1 and MAGE-C2 expressions were positively associated with high tumor grade and reduced recurrence-free survival; MAGE-C2 expression was also associated with tumor embolus and histologic type. 5-aza-CdR treatment alone could induce expression of MAGE-C2, whereas trichostatin A was able to synergistically enhance 5-aza-CdR-mediated MAGE-C2 transcription. MAGE-C1 and MAGE-C2 maybe potential targets for tumor immunotherapy, and their expressions are associated with advanced breast cancer and poor outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

    PubMed

    Piazzi, G; Selgrad, M; Garcia, M; Ceccarelli, C; Fini, L; Bianchi, P; Laghi, L; D'Angelo, L; Paterini, P; Malfertheiner, P; Chieco, P; Boland, C R; Bazzoli, F; Ricciardiello, L

    2013-04-30

    Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels. Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling.

  16. Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers

    PubMed Central

    Piazzi, G; Selgrad, M; Garcia, M; Ceccarelli, C; Fini, L; Bianchi, P; Laghi, L; D'Angelo, L; Paterini, P; Malfertheiner, P; Chieco, P; Boland, C R; Bazzoli, F; Ricciardiello, L

    2013-01-01

    Background: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. Methods: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2′-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. Results: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels. Conclusion: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling. PMID:23579212

  17. SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

    PubMed

    Almeida, Luciana O; Neto, Marinaldo P C; Sousa, Lucas O; Tannous, Maryna A; Curti, Carlos; Leopoldino, Andreia M

    2017-04-18

    Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

  18. Endothelial Cell–Specific Expression of Roundabout 4 Is Regulated by Differential DNA Methylation of the Proximal Promoter

    PubMed Central

    Okada, Yoshiaki; Funahashi, Nobuaki; Tanaka, Toru; Nishiyama, Yuji; Yuan, Lei; Shirakura, Keisuke; Turjman, Alexis S.; Kano, Yoshihiro; Naruse, Hiroki; Suzuki, Ayano; Sakai, Miki; Zhixia, Jiang; Kitajima, Kenji; Ishimoto, Kenji; Hino, Nobumasa; Kondoh, Masuo; Mukai, Yohei; Nakagawa, Shinsaku; García-Cardeña, Guillermo; Aird, William C.; Doi, Takefumi

    2017-01-01

    Objective The molecular basis of endothelial cell (EC)–specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5′-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. Methods and Results Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the −42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type–specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell–derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. Conclusions The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression. PMID:24855053

  19. Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

    PubMed

    Saulnier, Amandine; Vaissière, Thomas; Yue, Jiping; Siouda, Maha; Malfroy, Marine; Accardi, Rosita; Creveaux, Marion; Sebastian, Sinto; Shahzad, Naveed; Gheit, Tarik; Hussain, Ishraq; Torrente, Mariela; Maffini, Fausto Antonio; Calabrese, Luca; Chiesa, Fausto; Cuenin, Cyrille; Shukla, Ruchi; Fathallah, Ikbal; Matos, Elena; Daudt, Alexander; Koifman, Sergio; Wünsch-Filho, Victor; Menezes, Ana M B; Curado, Maria-Paula; Zaridze, David; Boffetta, Paolo; Brennan, Paul; Tommasino, Massimo; Herceg, Zdenko; Sylla, Bakary S

    2012-06-01

    The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy. Copyright © 2011 UICC.

  20. Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.

    PubMed

    Loughran, O; Malliri, A; Owens, D; Gallimore, P H; Stanley, M A; Ozanne, B; Frame, M C; Parkinson, E K

    1996-08-01

    We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.

  1. Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer.

    PubMed

    Smith, Heath A; Cronk, Robert J; Lang, Joshua M; McNeel, Douglas G

    2011-11-01

    Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer-testis antigens that are upregulated in MHC class I-deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus, SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study, we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines, but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably, SSX expression in patient tumor samples was restricted to metastatic lesions (5/22; 23%) and no expression was detected in primary prostate tumors examined (0/73; P < 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2-specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting.

  2. DNA promoter methylation-dependent transcription of the double C2-like domain β (DOC2B) gene regulates tumor growth in human cervical cancer.

    PubMed

    Kabekkodu, Shama Prasada; Bhat, Samatha; Radhakrishnan, Raghu; Aithal, Abhijit; Mascarenhas, Roshan; Pandey, Deeksha; Rai, Lavanya; Kushtagi, Pralhad; Mundyat, Gopinath Puthiya; Satyamoorthy, Kapaettu

    2014-04-11

    Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

  3. COX-2 expression is upregulated by DNA hypomethylation after hematopoietic stem cell transplantation.

    PubMed

    Domingo-Gonzalez, Racquel; Huang, Steven K; Laouar, Yasmina; Wilke, Carol A; Moore, Bethany B

    2012-11-01

    Hematopoietic stem cell transplant therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), after bone marrow transplantation (BMT) have impaired host defense against Gram-negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of PGE(2) by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE(2) from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized that epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5'-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared with control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) after treatment with 5-aza-2'-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 698-bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because TGF-β1 is elevated in lungs post-BMT, we tested whether TGF-β1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-β1-induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant-negative form of the TGF-βRII on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE(2) elevation post-BMT are due to hypomethylation of the COX-2 gene, which is at least partly regulated by TGF-β1.

  4. Association of NDRG1 gene promoter methylation with reduced NDRG1 expression in gastric cancer cells and tissue specimens.

    PubMed

    Chang, Xiaojing; Zhang, Shuanglong; Ma, Jinguo; Li, Zhenhua; Zhi, Yu; Chen, Jing; Lu, Yao; Dai, Dongqiu

    2013-05-01

    NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.

  5. Epigenetic regulation of the ras effector/tumour suppressor RASSF2 in breast and lung cancer.

    PubMed

    Cooper, W N; Dickinson, R E; Dallol, A; Grigorieva, E V; Pavlova, T V; Hesson, L B; Bieche, I; Broggini, M; Maher, E R; Zabarovsky, E R; Clark, G J; Latif, F

    2008-03-13

    RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.

  6. Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

    PubMed Central

    Horvat, Tomislav; Maglica, Željka; Vojta, Aleksandar; Zoldoš, Vlatka

    2014-01-01

    Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2′-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization. PMID:25093501

  7. Aberrant hypomethylation-mediated CD147 overexpression promotes aggressive tumor progression in human prostate cancer.

    PubMed

    Liang, Yu-Xiang; Mo, Ru-Jun; He, Hui-Chan; Chen, Jia-Hong; Zou, Jun; Han, Zhao-Dong; Lu, Jian-Ming; Cai, Chao; Zeng, Yan-Ru; Zhong, Wei-De; Wu, Chin-Lee

    2015-05-01

    Our previous study revealed the potential role of CD147 in human prostate cancer (PCa). Here, we investigated the CD147 promoter methylation status and the correlation with tumorigenicity in human PCa. CD147 mRNA and protein expression levels were both significantly higher in the 4 PCa cell lines, than in the 2 non-tumorigenic benign human prostatic epithelial cell lines (all P<0.01). We showed hypomethylation of promoter regions of CD147 in PCa cell lines with significant CD147 expression as compared to non-tumorigenic benign human prostatic epithelial cell lines slowly expressing CD147. Additionally, the treatment of methylated cell lines with 5-aza-2'-deoxycytidine increased CD147 expression significantly in low-expressing cell lines and also activated the expression of matrix metalloproteinase (MMP)-2, which may be one of the most important downstream targets of CD147. Furthermore, PCa tissues displayed decreased DNA methylation in the promoter region of CD147 compared to the corresponding non-cancerous prostate tissues, and methylation intensity correlated inversely with the CD147 mRNA levels. There was a significant negative correlation between CD147 mRNA levels and the number of methylated sites in PCa tissues (r=-0.467, P<0.01). In conclusion, our data offer convincing evidence for the first time that the DNA promoter hypomethylation of CD147 may be one of the regulatory mechanisms involved in the cancer-related overexpression of CD147 and may play a crucial role in the tumorigenesis of PCa.

  8. Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal transcriptional regulatory region in an antifolate-resistant HeLa cell line.

    PubMed

    Diop-Bove, Ndeye Khady; Wu, Julia; Zhao, Rongbao; Locker, Joseph; Goldman, I David

    2009-08-01

    This laboratory recently identified a novel proton-coupled folate transporter (PCFT) that mediates intestinal folate absorption and transport of folates into the central nervous system. The present study focuses on the definition of the minimum transcriptional regulatory region of this gene in HeLa cells and the mechanism(s) underlying the loss of PCFT expression in the methotrexate-resistant HeLa R1-11 cell line. The PCFT transcriptional regulatory controls were localized between -42 and +96 bases from the transcriptional start site using a luciferase-reporter gene system. The promoter is a G + C rich region of 139 nucleotides contained in a CpG island. HeLa R1-11 cells have no mutations in the PCFT open reading frame and its promoter; the transcription/translation machinery is intact because transient transfections in HeLa R1-11 and wild-type HeLa cells produced similar luciferase activities. Hypermethylation at CpG sites within the minimal transcriptional regulatory region was shown in HeLa R1-11 cells as compared with the parental PCFT-competent HeLa cells, using bisulfite conversion and sequence analysis. Treatment with 5-aza-2'-deoxycytidine resulted in a substantial restoration of transport and PCFT mRNA expression and small but significant decreases in methylation in the promoter region. In vitro methylation of the transfected reporter plasmid inhibited luciferase gene expression. Cytogenetics/fluorescence in situ hybridization indicated a loss of half the PCFT gene copies in HeLa R1-11 as compared with PCFT-competent HeLa cells. Taken together, promoter silencing through methylation and gene copy loss accounted for the loss of PCFT activity in antifolate-resistant HeLa R1-11 cells.

  9. Promoter hypermethylation profile of RASSF1A, FHIT, and sFRP1 in intracranial primitive neuroectodermal tumors.

    PubMed

    Chang, Qing; Pang, Jesse Chung-Sean; Li, Kay Ka Wai; Poon, Wai Sang; Zhou, Liangfu; Ng, Ho-Keung

    2005-12-01

    Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumors (SPNETs) are histologically alike intracranial PNETs found in different anatomical locations of the brain. Current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. The aim of this study was to investigate whether promoter hypermethylation of putative tumor suppressor genes was involved in both types of intracranial PNETs. We examined the methylation status at promoter regions of RASSF1A, FHIT, and sFRP1 by methylation-specific polymerase chain reaction in a cohort of 25 primary MBs, 9 primary SPNETs, and 3 MB and 2 SPNET cell lines. Our results revealed no promoter hypermethylation of RASSF1A, FHIT, and sFRP1 in 2 normal cerebellar and 5 normal cerebral tissue specimens examined. In contrast, promoter hypermethylation of RASSF1A was detected in 100% of primary MBs, 67% (6/9) of primary SPNETs, and all PNET cell lines. The frequency of promoter hypermethylation of RASSF1A was significantly lower in SPNETs than in MBs (Fisher exact test, P = .014). Treatment of RASSF1A-deficient PNET cell lines with 5-aza-2'deoxycytidine, a demethylating agent, restored RASSF1A expression, providing evidence that promoter hypermethylation contributes to transcriptional silencing. In addition, promoter hypermethylation of FHIT and sFRP1 was detected in 22% (2/9) and 11% (1/9) of SPNETs, respectively, but not in any MBs studied. In conclusion, our study demonstrates that promoter hypermethylation of RASSF1A is a common event in intracranial PNETs, whereas FHIT and sFRP1 are epigenetically affected in a fraction of SPNETs.

  10. Epigenetic regulation of proMMP-1 expression in the HT1080 human fibrosarcoma cell line.

    PubMed

    Poplineau, Mathilde; Dufer, Jean; Antonicelli, Frank; Trussardi-Regnier, Aurelie

    2011-06-01

    The matrix metalloproteinase (MMP) family members play an important role in various physiological and pathological processes. Although MMP-1 (collagenase-1) has been shown to be involved in tumor invasiveness, the regulation of its expression is still not fully elucidated and could implicate epigenetic mechanisms. The aim of this study was to analyze the effects of the Histone Deacetylase Inhibitor (HDI) trichostatin A (TSA) and the inhibitor of DNA methylation 5-aza-2'-deoxycytidine (5-azadC) on the proMMP-1 expression in the human HT1080 fibrosarcoma cell line. Real-time RT-PCR revealed that 5-azadC or 5-azadC + TSA but not TSA alone, despite global histone H4 hyperacetylation, increased proMMP-1 mRNA levels. This transcription activation was correlated with chromatin decondensation determined by nuclear texture image analysis technique. Western blot analysis of cell culture conditioned media revealed a significant increase in proMMP-1 secretion after 5-azadC or 5-azadC + TSA treatment compared to untreated cells. These results suggested that epigenetic mechanisms could be involved in proMMP-1 gene expression including chromatin supra-organization changes. Indeed, although the proMMP-1 gene promoter does not appear to contain CpG islands, its expression can be induced by the demethylating agent 5-azadC. Further experiments revealed that inhibition of protein neosynthesis by cycloheximide decreased 5-azadC-induced proMMP-1 mRNA, suggesting that epigenetically regulated intermediate molecules could be involved in proMMP-1 expression regulation in these cells.

  11. Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line

    PubMed Central

    Lacunza, Ezequiel; Garcia-Fabiani, Maria B.; Soler-Gerino, Mercedes C.; Cattaneo, Elizabeth R.; Quiroga, Ivana Y.; Abba, Martin C.; Coleman, Rosalind A.; Gonzalez-Baro, Maria R.

    2014-01-01

    The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells. PMID:24967918

  12. Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study.

    PubMed

    Fonteneau, M; Filliol, D; Anglard, P; Befort, K; Romieu, P; Zwiller, J

    2017-03-01

    DNA methylation is a major epigenetic process which regulates the accessibility of genes to the transcriptional machinery. In the present study, we investigated whether modifying the global DNA methylation pattern in the brain would alter cocaine intake by rats, using the cocaine self-administration test. The data indicate that treatment of rats with the DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (dAZA) and zebularine enhanced the reinforcing properties of cocaine. To obtain some insights about the underlying neurobiological mechanisms, a genome-wide methylation analysis was undertaken in the prefrontal cortex of rats self-administering cocaine and treated with or without dAZA. The study identified nearly 189 000 differentially methylated regions (DMRs), about half of them were located inside gene bodies, while only 9% of DMRs were found in the promoter regions of genes. About 99% of methylation changes occurred outside CpG islands. Gene expression studies confirmed the inverse correlation usually observed between increased methylation and transcriptional activation when methylation occurs in the gene promoter. This inverse correlation was not observed when methylation took place inside gene bodies. Using the literature-based Ingenuity Pathway Analysis, we explored how the differentially methylated genes were related. The analysis showed that increase in cocaine intake by rats in response to DNA methyltransferase inhibitors underlies plasticity mechanisms which mainly concern axonal growth and synaptogenesis as well as spine remodeling. Together with the Akt/PI3K pathway, the Rho-GTPase family was found to be involved in the plasticity underlying the effect of dAZA on the observed behavioral changes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  13. Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma.

    PubMed

    Suzuki, Fumihiko; Akahira, Jun-Ichi; Miura, Ikumi; Suzuki, Takashi; Ito, Kiyoshi; Hayashi, Shin-Ichi; Sasano, Hironobu; Yaegashi, Nobuo

    2008-12-01

    Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ERbeta in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ERbeta isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription-polymerase chain reaction were used to evaluate methylation status and expression of ERbeta isoforms. The relative abundance of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ERbeta5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ERbeta gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ERbeta1, ERbeta2, and ERbeta4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5-aza-2' deoxycytidine resulted in reexpression of the ERbeta gene. The results of our present study suggest that ERbeta is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of

  14. CHD5 a tumour suppressor is epigenetically silenced in hepatocellular carcinoma.

    PubMed

    Zhao, Rui; Wang, Nisha; Huang, Haili; Ma, Wenli; Yan, Qitao

    2014-07-01

    Chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a potent tumour suppressor by acting as a master regulator of a tumour-suppressive network. Its inactivation resulted from aberrant methylation in the promoter occurs in several types of human malignancy and is associated with malignant tumour behaviour. In human hepatocellular carcinoma (HCC), CHD5 gene expression, methylation status and tumour-suppressive function have not been elucidated. In this study, we focused on the epigenetic modification and tumour-suppressive mechanism of CHD5 gene in HCC. CHD5 expression in nine HCC cell lines and 30 pairs of HCC specimens and adjacent non-cancerous tissues were analysed by quantitative reverse transcription PCR and Western blotting. Methylation-specific sequencing and methylation-specific PCR were performed to examine DNA methylation status of the CHD5 promoter in HCC cell lines and samples. The effect of CHD5 restoration on proliferation, colony formation, senescence, apoptosis and tumourigenicity were examined. CHD5 expression was sinificantly down-regulated in HCC cell lines and tissues examined, and the -841 to -470 region of CHD5 promoter was hypermethylated in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine resulted in a striking regional demethylation of the -841 to -470 region of CHD5 promoter and an increase in CHD5 expression. The restoration of CHD5 expression inhibited tumour cell proliferation, colony formation and tumourigenicity and caused cellular senescence. Our findings demonstrate that CHD5 is a potential tumour suppressor gene epigenetically silenced in HCC. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression.

    PubMed

    Ushmorov, Alexey; Ritz, Olga; Hummel, Michael; Leithäuser, Frank; Möller, Peter; Stein, Harald; Wirth, Thomas

    2004-11-15

    Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of "crippling" mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells.

  16. EPB41L3, TSP-1 and RASSF2 as new clinically relevant prognostic biomarkers in diffuse gliomas

    PubMed Central

    Perez-Janices, Noemi; Blanco-Luquin, Idoia; Tuñón, Maria Teresa; Barba-Ramos, Edurne; Ibáñez, Berta; Zazpe-Cenoz, Idoya; Martinez-Aguillo, Maria Teresa; Hernandez, Berta; Martínez-Lopez, Enrique; Fernández, Agustin F.; Mercado, Maria Roasario; Cabada, Teresa; Escors, David; Megias, Diego; Guerrero-Setas, David

    2015-01-01

    Hypermethylation of tumor suppressor genes is one of the hallmarks in the progression of brain tumors. Our objectives were to analyze the presence of the hypermethylation of EPB41L3, RASSF2 and TSP-1 genes in 132 diffuse gliomas (astrocytic and oligodendroglial tumors) and in 10 cases of normal brain, and to establish their association with the patients’ clinicopathological characteristics. Gene hypermethylation was analyzed by methylation-specific-PCR and confirmed by pyrosequencing (for EPB41L3 and TSP-1) and bisulfite-sequencing (for RASSF2). EPB41L3, RASSF2 and TSP-1 genes were hypermethylated only in tumors (29%, 10.6%, and 50%, respectively), confirming their cancer-specific role. Treatment of cells with the DNA-demethylating-agent 5-aza-2′-deoxycytidine restores their transcription, as confirmed by quantitative-reverse-transcription-PCR and immunofluorescence. Immunohistochemistry for EPB41L3, RASSF2 and TSP-1 was performed to analyze protein expression; p53, ki-67, and CD31 expression and 1p/19q co-deletion were considered to better characterize the tumors. EPB41L3 and TSP-1 hypermethylation was associated with worse (p = 0.047) and better (p = 0.037) prognosis, respectively. This observation was confirmed after adjusting the results for age and tumor grade, the role of TSP-1 being most pronounced in oligodendrogliomas (p = 0.001). We conclude that EPB41L3, RASSF2 and TSP-1 genes are involved in the pathogenesis of diffuse gliomas, and that EPB41L3 and TSP-1 hypermethylation are of prognostic significance. PMID:25621889

  17. Sulfate Aerosols Promote Lung Cancer Metastasis by Epigenetically Regulating the Epithelial-to-Mesenchymal Transition (EMT).

    PubMed

    Yun, Yang; Gao, Rui; Yue, Huifeng; Guo, Lin; Li, Guangke; Sang, Nan

    2017-10-03

    Secondary inorganic aerosols (SIA), particularly sulfate aerosols, are central particulate matter (PM) constituents of severe haze formation in China and exert profound impacts on human health; however, our understanding of the mechanisms by which sulfate aerosols cause malignancy in lung carcinogenesis remains incomplete. Here, we show that exposure to secondary inorganic aerosols induced the invasion and migration of lung epithelial cells, and that (NH4)2SO4 exerted the most serious effects in vitro and promoted lung tumor metastasis in vivo. This action was associated with alterations of phenotype markers in the epithelial-to-mesenchymal transition (EMT), such as the up-regulation of fibronectin (Fn1) and the down-regulation of E-cadherin (E-cad). Hypoxia-inducible factor 1α (HIF-1α)-Snail signaling, regulated by the generation of reactive oxygen species (ROS), was involved in the (NH4)2SO4-induced EMT, and the potent antioxidant N-acetylcysteine (NAC) inhibited the activation of HIF-1α-Snail and blocked the EMT, cell invasion, and migration in response to (NH4)2SO4. Additionally, CpG hypermethylation in the E-cad promoter regions partly contributed to the (NH4)2SO4-regulated E-cad repression, and the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) restored the (NH4)2SO4-induced down-regulation of E-cad. Our findings reveal a potential mechanistic basis for exploring the association between sulfate aerosol exposure and increased malignancy during lung carcinogenesis, and suggest new approaches for the treatment, improvement, and prevention of lung cancer resulting from sulfate aerosol exposure in severe haze-fog.

  18. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    PubMed

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

  19. Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro and In Vivo

    PubMed Central

    Ye, Danna; Li, Tong; Heraud, Philip; Parnpai, Rangsun

    2016-01-01

    Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiation in vitro and in vivo was determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiation in vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCs in vitro. Their in vivo function needs further investigation. PMID:27242905

  20. A role for TET2 in parathyroid carcinoma.

    PubMed

    Barazeghi, Elham; Gill, Anthony J; Sidhu, Stan; Norlén, Olov; Dina, Roberto; Palazzo, F Fausto; Hellman, Per; Stålberg, Peter; Westin, Gunnar

    2017-07-01

    Primary hyperparathyroidism (pHPT) is rarely caused by parathyroid carcinoma (PC, <1-5% of pHPT cases). The TET proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and inactivation by mutation or epigenetic deregulation of TET1 and TET2 play important roles in various cancers. Recently, we found that 5hmC was severely reduced in all of the analyzed PCs and with deranged expression of TET1 for the majority of PCs. Here, we have examined the expression of the TET2 protein in 15 5hmC-negative PCs from patients who had local invasion or metastases. Cell growth and cell migratory roles for TET2 as well as epigenetic deregulated expression were addressed. Immunohistochemistry revealed very low/undetectable expression of TET2 in all PCs and verified for two PCs that were available for western blotting analysis. Knockdown of TET2 in the parathyroid cell line sHPT-1 resulted in increased cell growth and increased cell migration. DNA sequencing of TET2 in PCs revealed two common variants and no obvious inactivating mutations. Quantitative bisulfite pyrosequencing analysis of the TET2 promoter CpG island revealed higher CpG methylation level in the PCs compared to that in normal tissues and treatment of a PC primary cell culture with the DNA methylation inhibitor 5-aza-2'-deoxycytidine caused increased expression of the methylated TET2 gene. Hence, the data suggest that deregulated expression of TET2 by DNA hypermethylation may contribute to the aberrantly low level of 5hmC in PCs and further that TET2 plays a cell growth and cell migratory regulatory role and may constitute a parathyroid tumor suppressor gene. © 2017 Society for Endocrinology.

  1. Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

    SciTech Connect

    Liu Wenbin; Cui Zhihong; Ao Lin; Zhou Ziyuan; Zhou Yanhong; Yuan Xiaoyan; Xiang Yunlong; Liu Jinyi Cao Jia

    2011-02-15

    To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.

  2. Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro and In Vivo.

    PubMed

    Ye, Danna; Li, Tong; Heraud, Philip; Parnpai, Rangsun

    2016-01-01

    Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiation in vitro and in vivo was determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiation in vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCs in vitro. Their in vivo function needs further investigation.

  3. The roles of BTG3 expression in gastric cancer: a potential marker for carcinogenesis and a target molecule for gene therapy.

    PubMed

    Gou, Wen-feng; Yang, Xue-feng; Shen, Dao-fu; Zhao, Shuang; Liu, Yun-peng; Sun, Hong-zhi; Takano, Yasuo; Su, Rong-jian; Luo, Jun-sheng; Zheng, Hua-chuan

    2015-08-14

    BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis and angiogenesis, cell cycle progression, and induce differentiation in various cells. Here, we found that BTG3 overexpression inhibited proliferation, induced S/G2 arrest, differentiation, autophagy, apoptosis, suppressed migration and invasion in MKN28 and MGC803 cells (p < 0.05). BTG3 transfectants showed a higher mRNA expression of p27, Bax, 14-3-3, Caspase-3, Caspase-9, Beclin 1, NF-κB, IL-1, -2, -4, -10 and -17, but a lower mRNA expression of p21, MMP-9 and VEGF than the control and mock (p < 0.05). At protein level, BTG3 overexpression increased the expression of CDK4, AIF, LC-3B, Beclin 1 and p38 (p < 0.05), but decreased the expression of p21 and β-catenin in both transfectants (p < 0.05). After treated with cisplatin, MG132, paclitaxel and SAHA, both BTG3 transfectants showed lower viability and higher apoptosis than the control in both time- and dose-dependent manners (p < 0.05). BTG3 expression was restored after 5-aza-2'-deoxycytidine or MG132 treatment in gastric cancer cells. BTG3 expression was decreased in gastric cancer in comparison to the adjacent mucosa (p < 0.05), and positively correlated with venous invasion and dedifferentiation of cancer (p < 0.05). It was suggested that BTG3 expression might contribute to gastric carcinogenesis. BTG3 overexpression might reverse the aggressive phenotypes and be employed as a potential target for gene therapy of gastric cancer.

  4. Rb silencing mediated by the down-regulation of MeCP2 is involved in cell transformation induced by long-term exposure to hydroquinone.

    PubMed

    Liu, Linhua; Ling, Xiaoxuan; Wu, Minhua; Chen, Jialong; Chen, Shaoqiao; Tan, Qiang; Chen, Jiansong; Liu, Jiaxian; Zou, Fei

    2017-02-01

    Hydroquinone (HQ), a metabolite of benzene, is a well-known human carcinogen; however, its molecular mechanisms of action remain unclear. MeCP2 has been traditionally described as a transcriptional repressor, though growing evidence indicates that it also activates gene expression. Here, we investigated whether some epigenetic machinery genes are aberrantly expressed as target tumor suppressor genes in HQ-transformed TK6 lymphoblastoid cells. Our results showed that treatment with 5-Aza-2'-deoxycytidine or trichostatin A enhanced the expression of Rb, resulting in cell arrest in G1-phase, and subsequently, an increase in apoptosis and a decrease in cell growth. Moreover, we hypothesised that Rb was silenced by the down-regulation of MeCP2 in HQ-transformed cells, resulting in the dynamic expression of Rb and epigenetic machinery proteins in HQ-transformed cells at different time points. The expression of Rb and MeCP2 in patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed that positive staining for MeCP2 or Rb was significantly lower in B-NHL tumor tissues, and these changes were significantly and negatively correlated with the grade of B-NHL. The restoration of MeCP2 in HQ-transformed cells enhanced the expression of Rb, promoted cell apoptosis, and inhibited cell growth. The changes in the expression patterns of MeCP2 and Rb were inversely correlated with the degree of DNA methylation. A ChiP assay revealed that MeCP2 proteins were recruited to the Rb promoter with lower 5'-methylcytosine levels. In conclusion, we demonstrated that the down-regulation of MeCP2 silences Rb, a process involved in cell transformation resulting from long-term exposure to HQ. © 2016 Wiley Periodicals, Inc.

  5. Epigenetic Modification of TLRs in Leukocytes Is Associated with Increased Susceptibility to Salmonella enteritidis in Chickens

    PubMed Central

    Zhao, Guiping; Zheng, Maiqing; Li, Peng; Wang, Huihua; Zhu, Yun; Chen, Jilan; Wen, Jie

    2012-01-01

    Toll-like receptors (TLRs) signaling pathways are the first lines in defense against Salmonella enteritidis (S. enteritidis) infection but the molecular mechanism underlying susceptibility to S. enteritidis infection in chicken remains unclear. SPF chickens injected with S. enteritidis were partitioned into two groups, one consisted of those from Salmonella-susceptible chickens (died within 5 d after injection, n = 6), the other consisted of six Salmonella-resistant chickens that survived for 15 d after injection. The present study shows that the bacterial load in susceptible chickens was significantly higher than that in resistant chickens and TLR4, TLR2-1 and TLR21 expression was strongly diminished in the leukocytes of susceptible chickens compared with those of resistant chickens. The induction of expression of pro-inflammatory cytokine genes, IL-6 and IFN-β, was greatly enhanced in the resistant but not in susceptible chickens. Contrasting with the reduced expression of TLR genes, those of the zinc finger protein 493 (ZNF493) gene and Toll-interacting protein (TOLLIP) gene were enhanced in the susceptible chickens. Finally, the expression of TLR4 in peripheral blood mononuclear cells (PBMCs) infected in vitro with S. enteritidis increased significantly as a result of treatment with 5-Aza-2-deoxycytidine (5-Aza-dc) while either 5-Aza-dc or trichostatin A was effective in up-regulating the expression of TLR21 and TLR2-1. DNA methylation, in the predicted promoter region of TLR4 and TLR21 genes, and an exonic CpG island of the TLR2-1 gene was significantly higher in the susceptible chickens than in resistant chickens. Taken together, the results demonstrate that ZNF493-related epigenetic modification in leukocytes probably accounts for increased susceptibility to S. enteritidis in chickens by diminishing the expression and response of TLR4, TLR21 and TLR2-1. PMID:22438967

  6. Epigenetic regulation of HGF/Met receptor axis is critical for the outgrowth of bone metastasis from breast carcinoma.

    PubMed

    Bendinelli, Paola; Maroni, Paola; Matteucci, Emanuela; Desiderio, Maria Alfonsina

    2017-02-02

    Our translational research deals with the influence of microenvironment on the phenotype and colonization of bone metastases from breast carcinoma, and on pre-metastatic niche formation. The aim of the present study was to clarify the origin of hepatocyte growth factor (HGF), ligand of Met receptor, the control of the axis HGF/Met by DNA methylation, and its importance for the nexus supportive cells-metastatic cells and for metastasis outgrowth. In bone metastasis of the 1833-xenograft model, DNA methyltransferase blockade using the chemotherapic drug 5-aza-2'-deoxycytidine (decitabine) strongly reduced the expression of HGF/Met receptor axis and of E-cadherin, with decrease of metastasis wideness and osteolysis, prolonging mice survival. Thus, DNA methylation events acted as commanders of breast carcinoma cells metastatizing to bone influencing the epithelial phenotype. HGF emerged as a bone-marrow stimulus, and the exosomes seemed to furnish HGF to metastatic cells. In fact, decitabine treatment similarly affected some markers of these microvesicles and HGF, indicating that its supply to recipient cells was prevented. Notably, in bone metastasis the hypomethylation of HGF, Met and E-cadherin promoters did not appear responsible for their elevated expression, but we suggest the involvement of hypermethylated regulators and of Wwox oncosuppressor, the latter being affected by decitabine. Wwox expression increased under decitabine strongly localizing in nuclei of bone metastases. We hypothesize a role of Wwox in Met activity since in vitro Wwox overexpression downregulated the level of nuclear-Met protein fragment and Met stability, also under long exposure of 1833 cells to decitabine. HGF enhanced phosphoMet and the activity in nuclei, an effect partially prevented by decitabine. Altogether, the data indicated the importance to target the tumor microenvironment by blocking epigenetic mechanisms, which control critical events for colonization such as HGF/Met axis

  7. Epigenetic regulation of CFTR in salivary gland.

    PubMed

    Shin, Yong-Hwan; Lee, Sang-Woo; Kim, Minkyoung; Choi, Se-Young; Cong, Xin; Yu, Guang-Yan; Park, Kyungpyo

    2016-12-02

    Cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in exocrine secretion, including salivary glands. However, its functional expression in salivary glands has not been rigorously studied. In this study, we investigated the expression pattern and regulatory mechanism of CFTR in salivary glands using immunohistochemistry, western blot analysis, Ussing chamber study, methylation-specific PCR, and bisulfite sequencing. Using an organ culture technique, we found that CFTR expression was first detected on the 15th day at the embryonic stage (E15) and was observed in ducts but not in acini. CFTR expression was confirmed in HSG and SIMS cell lines, which both originated from ducts, but not in the SMG C-6 cell line, which originated from acinar cells. Treatment of SMG C-6 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the expression level of CFTR mRNA in a time-dependent manner. Restoration of CFTR was further confirmed by a functional study. In the Ussing chamber study, 10 μM Cact-A1, a CFTR activator, did not evoke any currents in SMG C-6 cells. In contrast, in SMG C-6 cells pretreated with 5-Aza-CdR, Cact-A1 evoked a robust increase of currents, which were inhibited by the CFTR inhibitor CFTRinh-172. Furthermore, forskolin mimicked the currents activated by Cact-A1. In our epigenetic study, SMG C-6 cells showed highly methylated CG pairs in the CFTR CpG island and most of the methylated CG pairs were demethylated by 5-Aza-CdR. Our results suggest that epigenetic regulation is involved in the development of salivary glands by silencing the CFTR gene in a tissue-specific manner.

  8. Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

    PubMed

    Maugeri, Marco; Barbagallo, Davide; Barbagallo, Cristina; Banelli, Barbara; Di Mauro, Stefania; Purrello, Francesco; Magro, Gaetano; Ragusa, Marco; Di Pietro, Cinzia; Romani, Massimo; Purrello, Michele

    2016-12-13

    Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5'-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5'-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

  9. ‘Default’ generated neonatal regulatory T cells are hypomethylated at conserved non-coding sequence 2 and promote long-term cardiac allograft survival

    PubMed Central

    Cheng, Chao; Wang, Sihua; Ye, Ping; Huang, Xiaofan; Liu, Zheng; Wu, Jie; Sun, Yuan; Xie, Aini; Wang, Guohua; Xia, Jiahong

    2014-01-01

    Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4+ T cells have an intrinsic ‘default’ mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3gfp mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2−/− mice with allograft cardiac transplantation and transferred with syngeneic CD4+ effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4+ T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0·001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2′-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4+ T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the ‘default’ pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection. PMID:24944101

  10. BTG1 expression correlates with pathogenesis, aggressive behaviors and prognosis of gastric cancer: a potential target for gene therapy.

    PubMed

    Zheng, Hua-chuan; Li, Jing; Shen, Dao-fu; Yang, Xue-feng; Zhao, Shuang; Wu, Ya-zhou; Takano, Yasuo; Sun, Hong-zhi; Su, Rong-jian; Luo, Jun-sheng; Gou, Wen-feng

    2015-08-14

    Here, we found that BTG1 overexpression inhibited proliferation, migration and invasion, induced G2/M arrest, differentiation, senescence and apoptosis in BGC-823 and MKN28 cells (p < 0.05). BTG1 transfectants showed a higher mRNA expression of Cyclin D1 and Bax, but a lower mRNA expression of cdc2, p21, mTOR and MMP-9 than the control and mock (p < 0.05). After treated with cisplatin, MG132, paclitaxel and SAHA, both BTG1 transfectants showed lower mRNA viability and higher apoptosis than the control in both time- and dose-dependent manners (p < 0.05) with the hypoexpression of chemoresistance-related genes (slug, CD147, GRP78, GRP94, FBXW7 TOP1, TOP2 and GST-π). BTG1 expression was restored after 5-aza-2'-deoxycytidine treatment in gastric cancer cells. BTG1 expression was statistically lower in gastric cancer than non-neoplastic mucosa and metastatic cancer in lymph node (p < 0.05). BTG1 expression was positively correlated with depth of invasion, lymphatic and venous invasion, lymph node metastasis, TNM staging and worse prognosis (p < 0.05). The diffuse-type carcinoma showed less BTG1 expression than intestinal- and mixed-type ones (p < 0.05). BTG1 overexpression suppressed tumor growth and lung metastasis of gastric cancer cells by inhibiting proliferation, enhancing autophagy and apoptosis in xenograft models. It was suggested that down-regulated BTG1 expression might promote gastric carcinogenesis partially due to its promoter methylation. BTG1 overexpression might reverse the aggressive phenotypes and be employed as a potential target for gene therapy of gastric cancer.

  11. Epigenetic modulation of the biophysical properties of drug-resistant cell lipids to restore drug transport and endocytic functions.

    PubMed

    Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Lu, Shan; Labhasetwar, Vinod

    2012-09-04

    In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

  12. Metabolomic profiling reveals potential markers and bioprocesses altered in bladder cancer progression.

    PubMed

    Putluri, Nagireddy; Shojaie, Ali; Vasu, Vihas T; Vareed, Shaiju K; Nalluri, Srilatha; Putluri, Vasanta; Thangjam, Gagan Singh; Panzitt, Katrin; Tallman, Christopher T; Butler, Charles; Sana, Theodore R; Fischer, Steven M; Sica, Gabriel; Brat, Daniel J; Shi, Huidong; Palapattu, Ganesh S; Lotan, Yair; Weizer, Alon Z; Terris, Martha K; Shariat, Shahrokh F; Michailidis, George; Sreekumar, Arun

    2011-12-15

    Although alterations in xenobiotic metabolism are considered causal in the development of bladder cancer, the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in bladder cancer. This metabolic signature distinguished both normal and benign bladder from bladder cancer. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing bladder cancer from controls and also nonmuscle from muscle-invasive bladder cancer. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in bladder cancer. In particular, we validated tumor-associated hypermethylation in the cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) promoters of bladder cancer tissues by bisulfite sequence analysis and methylation-specific PCR and also by in vitro treatment of T-24 bladder cancer cell line with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of bladder cancer specimens compared with matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of bladder cancer, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.

  13. Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells.

    PubMed

    Fan, Huitao; Zhang, Rui; Tesfaye, Dawit; Tholen, Ernst; Looft, Christian; Hölker, Michael; Schellander, Karl; Cinar, Mehmet Ulas

    2012-12-01

    Satellite cells function as skeletal muscle stem cells to support postnatal muscle growth and regeneration following injury or disease. There is great promise for the improvement of muscle performance in livestock and for the therapy of muscle pathologies in humans by the targeting of myostatin (MSTN) in this cell population. Human diet contains many histone deacetylase (HDAC) inhibitors, such as the bioactive component sulforaphane (SFN), whose epigenetic effects on MSTN gene in satellite cells are unknown. Therefore, we aimed to investigate the epigenetic influences of SFN on the MSTN gene in satellite cells. The present work provides the first evidence, which is distinct from the effects of trichostatin A (TSA), that SFN supplementation in vitro not only acts as a HDAC inhibitor but also as a DNA methyltransferase (DNMT) inhibitor in porcine satellite cells. Compared with TSA and 5-aza-2'-deoxycytidine (5-aza-dC), SFN treatment significantly represses MSTN expression, accompanied by strongly attenuated expression of negative feedback inhibitors of the MSTN signaling pathway. miRNAs targeting MSTN are not implicated in posttranscriptional regulation of MSTN. Nevertheless, a weakly enriched myoblast determination (MyoD) protein associated with diminished histone acetylation in the MyoD binding site located in the MSTN promoter region may contribute to the transcriptional repression of MSTN by SFN. These findings reveal a new mode of epigenetic repression of MSTN by the bioactive compound SFN. This novel pharmacological, biological activity of SFN in satellite cells may thus allow for the development of novel approaches to weaken the MSTN signaling pathway, both for therapies of human skeletal muscle disorders and for livestock production improvement.

  14. Development of TRAIL Resistance by Radiation-Induced Hypermethylation of DR4 CpG Island in Recurrent Laryngeal Squamous Cell Carcinoma

    SciTech Connect

    Lee, Jong Cheol; Lee, Won Hyeok; Min, Young Joo; Cha, Hee Jeong; Han, Myung Woul; Chang, Hyo Won; Kim, Sun-A; Choi, Seung-Ho; Kim, Seong Who; Kim, Sang Yoon

    2014-04-01

    Purpose: There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis. Methods and Materials: Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line. Results: HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2′-deoxycytidine. Conclusion: Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

  15. Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced Pathogenicities.

    PubMed

    Yu, Jing-Yi; He, Xiao-Long; Puthiyakunnon, Santhosh; Peng, Liang; Li, Yan; Wu, Li-Sha; Peng, Wen-Ling; Zhang, Ya; Gao, Jie; Zhang, Yao-Yuan; Boddu, Swapna; Long, Min; Cao, Hong; Huang, Sheng-He

    2015-10-01

    Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli-induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl- L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli-caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5- Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli-induced pathogenicities in a manner dependent on MUC2.

  16. Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

    PubMed Central

    Hao, Xiao-Wen; Zhu, Sheng-Tao; He, Yuan-Long; Li, Peng; Wang, Yong-Jun; Zhang, Shu-Tian

    2012-01-01

    AIM: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis. METHODS: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro. RESULTS: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2’-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3

  17. Formation of Fused-Ring 2′-Deoxycytidine Adducts from 1-Chloro-3-buten-2-one, an in Vitro 1,3-Butadiene Metabolite, under in Vitro Physiological Conditions

    PubMed Central

    Sun, Liang; Pelah, Avishay; Zhang, Dong-Ping; Zhong, Yu-Fang; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu; Elfarra, Adnan A.

    2013-01-01

    1-Chloro-3-buten-2-one (CBO) is a potential metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO is a bifunctional alkylating agent that readily reacts with glutathione (GSH) to form mono-GSH and di-GSH adducts. Recently, CBO and its precursor 1-chloro-2-hydroxy-3-butene (CHB) were found to be cytotoxic and genotoxic in human liver cells in culture with CBO being approximately 100-fold more potent than CHB. In the present study, CBO was shown to react readily with 2′-deoxycytidine (dC) under in vitro physiological conditions (pH 7.4, 37 °C) to form four dC adducts with the CBO moieties forming fused rings with the N3 and N4 atoms of dC. The four products were structurally characterized as 2-hydroxy-2-hydroxymethyl-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahy dro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-1 and dC-2, a pair of diastereomers), 4-chloromethyl-4-hydroxy-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahydr o-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-3), and 2-chloromethyl-2-hydroxy-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahydr o-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-4). Interestingly, dC-1 and dC-2 were stable under our experimental conditions (pH 7.4, 37 °C, 6 h) and existed in equilibrium as indicated by HPLC analysis, whereas dC-3 and dC-4 were labile with the half-lives being 3.0 ± 0.36 and 1.7 ± 0.06 h, respectively. Decomposition of dC-4 produced both dC-1 and dC-2, whereas acid hydrolysis of dC-1/dC-2 and dC-4 in 1 M HCl at 100 °C for 30 min yielded the deribosylated adducts dC-1H/dC-2H and dC-4H, respectively. Because fused-ring dC adducts of other chemicals are mutagenic, the characterized CBO-dC adducts could be mutagenic and play a role in the cytotoxicity and genotoxicity of CBO and its precursors, CHB and BD. The CBO-dC adducts may also be used as standards to characterize CBO-DNA adducts and to develop potential biomarkers for CBO formation in vivo. PMID:24020501

  18. Ski is silenced by methylation and acts as tumor suppressor in non-small cell lung cancer.

    PubMed

    Xie, Mian; Wu, Xiaojun; He, Chaosheng; Zhang, Jiexia; Zhang, Jinjun

    2015-12-12

    Epigenetic silencing of tumor suppressors contributes to the development and progression of lung cancer. We recently found that Ski was hypermethylated in lung cancer. This study aimed to clarify its epigenetic alteration, molecular mechanisms and clinical significance in lung cancer. Ski methylation was evaluated by methylation-specific PCR (MS-PCR) and bisulfite sequencing. mRNA level of Ski was measured by RT-PCR and compared with the methylation status. Ski methylation correlated with decreased mRNA expression in human lung cancer cell lines. Ski hypermethylation was detected in 56.0% of primary lung tumors and associated with poor differentiation and late tumor stage. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-2'dC) restored Ski expression. Re-expression of Ski in lung cancer cells inhibited cell proliferation, clonogenicity, migration, invasion and tumor formation. Ski decreased transcriptional activities of Smads and TAZ. Multivariate analysis showed that patients with Ski positive expression had a better overall survival in resected non-small cell lung cancer (NSCLC) patients. Our results revealed that Ski acts as a tumor suppressor inactivated by DNA methylation and is an independent prognostic factor of lung cancer.

  19. Myogenic-induced mesenchymal stem cells are capable of modulating the immune response by regulatory T cells

    PubMed Central

    Joo, Sunyoung; Lim, Hyun Ju; Jackson, John D; Atala, Anthony

    2014-01-01

    Cell therapy for patients who have intractable muscle disorders may require highly regenerative cells from young, healthy allogeneic donors. Mesenchymal stem cells are currently under clinical investigation because they are known to induce muscle regeneration and believed to be immune privileged, thus making them suitable for allogeneic applications. However, it is unclear whether allogeneic and myogenic-induced mesenchymal stem cells retain their immunomodulatory characteristics. Therefore, our aim was to evaluate the effects of mesenchymal stem cell differentiation on the immune characteristics of cells in vitro. We investigated the immunologic properties of mesenchymal stem cells after myogenic induction. Mesenchymal stem cells were obtained from C57BL/6 mice and the C3H/10T1/2 murine mesenchymal stem cell line. Two different 5-aza-2′-deoxycytidine doses (0.5 and 3 µM) were evaluated for their effects on mesenchymal stem cell skeletal myogenic differentiation potential, immune antigen expression, and mixed lymphocytic reactions. Using a mixed lymphocytic reaction, we determined the optimal splenocyte proliferation inhibition dose. The induction of regulatory T cells was markedly increased by the addition of 3 µM 5-aza-2′-deoxycytidine–treated mesenchymal stem cells. Myogenic-induced mesenchymal stem cells do not elicit alloreactive lymphocyte proliferative responses and are able to modulate immune responses. These findings support the hypothesis that myogenic-induced mesenchymal stem cells may be transplantable across allogeneic barriers. PMID:24555015

  20. Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

    SciTech Connect

    Merlevede, Jane; Droin, Nathalie; Qin, Tingting; Meldi, Kristen; Yoshida, Kenichi; Morabito, Margot; Chautard, Emilie; Auboeuf, Didier; Fenaux, Pierre; Braun, Thorsten; Itzykson, Raphael; de Botton, Stephane; Quesnel, Bruno; Commes, Therese; Jourdan, Eric; Vainchenker, William; Bernard, Olivier; Pata-Merci, Noemie; Solier, Stephanie; Gayevskiy, Velimir; Dinger, Marcel E.; Cowley, Mark J.; Selimoglu-Buet, Dorothee; Meyer, Vincent; Artiguenave, Francois; Deleuze, Jean -Francois; Preudhomme, Claude; Stratton, Michael R.; Alexandrov, Ludmil B.; Padron, Eric; Ogawa, Seishi; Koscielny, Serge; Figueroa, Maria; Solary, Eric

    2016-02-24

    The cytidine analogues azacytidine and 5-aza-2’-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14 ± 5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Lastly, our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.

  1. Drug-loaded biodegradable microspheres for image-guided combinatory epigenetic therapy in cells

    NASA Astrophysics Data System (ADS)

    Xu, Ronald X.; Xu, Jeff S.; Zuo, Tao; Shen, Rulong; Huang, Tim H.; Tweedle, Michael F.

    2011-02-01

    We synthesize drug-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres for image-guided combinatory epigenetic therapy in MCF-10A human mammary epithelial cells. LY294002 and Nile Red are encapsulated in microspheres for sustained drug release and fluorescence microscopic imaging. Drug-loaded microspheres target MCF-10A cells through a three-step binding process involving biotinylated antibody, streptavidin, and biotinylated microspheres. LY294002 loaded microspheres and 5-Aza-2-deoxycytidine are applied to MCF-10A cells for combinatory PI3K/AKT inhibition and deoxyribonucleic acid (DNA) demethylation. Our study implies the technical potential of disease targeting and image-guided combinatory epigenetic therapy using drug-loaded multifunctional biodegradable PLGA microspheres.

  2. DNA methylation controls unmethylated transcription start sites in the genome in trans.

    PubMed

    Cheishvili, David; Christiansen, Steffan; Stochinsky, Rebecca; Pepin, Anne-Sophie; Sapozhnikov, Daniel M; Zhou, Rudy; Schmeltzer, Lauren; Dymov, Sergey; Szyf, Moshe

    2017-05-01

    DNA methylation downregulates transcription. However, a large number of genes, which are unmethylated in the promoter region, are inactive. We tested the hypothesis that these genes are regulated by DNA methylation of upstream regulators. We inhibited DNMT1 with 5-aza-2'-deoxycytidine or depleted it with shRNA to map the transcription initiation positions controlled by DNMT1 using ChIPseq with RNApolIIser5 antibody. Ingenuity pathway analysis identified potential methylated upstream regulators. Their functional role in controlling unmethylated promoters was determined by CRISPR/Cas9 gene editing. We show that a large group of unmethylated promoters is regulated by DNMT1 through DNA methylation dependent silencing of upstream regulators such as transcription factor HNF4A. The landscape of genes regulated by DNA methylation is more wide-ranging than genes downregulated by methylation of their own cis-regulatory sequences; regulation of unmethylated promoters is dependent on the methylation state of upstream trans regulators.

  3. Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

    PubMed Central

    Merlevede, Jane; Droin, Nathalie; Qin, Tingting; Meldi, Kristen; Yoshida, Kenichi; Morabito, Margot; Chautard, Emilie; Auboeuf, Didier; Fenaux, Pierre; Braun, Thorsten; Itzykson, Raphael; de Botton, Stéphane; Quesnel, Bruno; Commes, Thérèse; Jourdan, Eric; Vainchenker, William; Bernard, Olivier; Pata-Merci, Noemie; Solier, Stéphanie; Gayevskiy, Velimir; Dinger, Marcel E.; Cowley, Mark J.; Selimoglu-Buet, Dorothée; Meyer, Vincent; Artiguenave, François; Deleuze, Jean-François; Preudhomme, Claude; Stratton, Michael R.; Alexandrov, Ludmil B.; Padron, Eric; Ogawa, Seishi; Koscielny, Serge; Figueroa, Maria; Solary, Eric

    2016-01-01

    The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect. PMID:26908133

  4. FT-IR Microspectrometry Reveals the Variation of Membrane Polarizability due to Epigenomic Effect on Epithelial Ovarian Cancer

    PubMed Central

    Hsu, Morris M. H.; Huang, Pei-Yu; Lee, Yao-Chang; Fang, Yuang-Chuen; Chan, Michael W. Y.; Lee, Cheng-I

    2014-01-01

    Ovarian cancer, as well as other cancers, is primarily caused by methylation at cytosines in CpG islands, but the current marker for ovarian cancer is low in sensitivity and failed in early-stage detection. Fourier transform infrared (FT-IR) spectroscopy is powerful in analysis of functional groups within molecules, and infrared microscopy illustrates the location of specific groups within single cells. In this study, we applied HPLC and FT-IR microspectrometry to study normal epithelial ovarian cell line immortalized ovarian surface epithelium (IOSE), two epithelial ovarian cell lines (A2780 and CP70) with distinct properties, and the effect of a cancer drug 5-aza-2'-deoxycytidine (5-aza) without labeling. Our results reveal that inhibition of methylation on cytosine with 5-aza initiates the protein expression. Furthermore, paraffin-adsorption kinetic study allows us to distinguish hypermethylated and hypomethyated cells, and this assay can be a potential diagnosis method for cancer screening. PMID:25299694

  5. Differential 14-3-3 sigma DNA methylation and expression in c-myc- and activated H-ras-transformed cells under r- and K-selection.

    PubMed

    Sato, Hiroyuki; Nakamura, Yukari; Motokura, Toru

    2006-05-08

    We cloned rat 14-3-3 sigma, a mediator of p53 tumor suppressor, as a target of K-selection. 14-3-3 sigma expression is suppressed with DNA methylation in breast cancers while its overexpression with hypomethylation is frequent in pancreatic cancers. These opposite findings were recapitulated through r- and K-selection of transformed rat embryo fibroblasts. 14-3-3 sigma expression was suppressed with DNA methylation after r-selection and the gene was overexpressed and demethylated in K-selected cells. 5-aza-2'-deoxycytidine recovered 14-3-3 sigma expression in r-selected cells. The presence of heterogeneous methylation patterns and expression levels before selection suggests that different 14-3-3 sigma expression levels play a role as a prerequisite for selection and clonal evolution.

  6. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles.

    PubMed

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2'-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2'-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs.

  7. Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations

    PubMed Central

    Kaneno, Ramon; Shurin, Galina V; Tourkova, Irina L; Shurin, Michael R

    2009-01-01

    The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations. PMID:19591684

  8. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

    PubMed Central

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2′-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2′-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs. PMID:26366077

  9. Enhancement of chemotherapeutic efficacy in hypermethylator breast cancer cells through targeted and pharmacologic inhibition of DNMT3b.

    PubMed

    Sandhu, Rupninder; Rivenbark, Ashley G; Coleman, William B

    2012-01-01

    A subset of primary breast cancers and breast cancer cell lines express a hypermethylation defect (characterized by DNMT hyperactivity and DNMT3b overexpression) which contributes to chemotherapy resistance and provides a target for development of new treatment strategies. The objective of the current study was to determine if targeting the epigenome enhances the sensitivity of breast cancer cells to cytotoxic chemotherapy. Hypermethylator breast cancer cell lines (MDA-MB-453, BT549, and Hs578T) were treated with 250 or 500 nM 5-aza-2'-deoxycytidine (5-aza) and/or were subjected to RNAi-mediated DNMT3b knockdown (KD), and then tested for sensitivity to doxorubicin hydrochloride (DOX), paclitaxel (PAX), and 5-fluorouracil (5-FU). In MDA-MB-453 cells, DNMT3b KD reduces the IC(50) for DOX from 0.086 to 0.048 μM (44% reduction), for PAX from 0.497 to 0.376 nM (24%), and for 5-FU from 0.817 to 0.145 mM (82%). Treatment with 250 nM 5-aza for 7 days did not increase the efficacy of DOX, PAX, or 5-FU, but 7-day treatment with 500 nM 5-aza sensitized cells, reducing the IC(50) for DOX to 0.035 μM (60%), PAX to 0.311 nM (37%), and 5-FU to 0.065 mM (92%). 5-aza treatment of DNMT3b KD cells reduced the IC(50) for DOX to 0.036 μM (59%), for PAX to 0.313 nM (37%) and for 5-FU to 0.067 (92%). Similar trends of enhancement of cell kill were seen in BT549 (13-60%) and Hs578T (29-70%) cells after RNAi-mediated DNMT3b KD and/or treatment with 5-aza. The effectiveness of DOX, PAX, and 5-FU is enhanced through targeted and/or pharmacological inhibition of DNMT3b, strongly suggesting that combined epigenetic and cytotoxic treatment will improve the efficacy of breast cancer chemotherapy.

  10. Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids

    PubMed Central

    Raghavan, Vijay; Vijayaraghavalu, Sivakumar; Peetla, Chiranjeevi; Yamada, Masayoshi; Morisada, Megan; Labhasetwar, Vinod

    2016-01-01

    Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study’s objective was to determine the effects of the epigenetic drug 5-aza-2′-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of Cholesterol-Sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lβ] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport

  11. Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells.

    PubMed

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2009-11-01

    In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect

  12. A Novel Combinatorial Epigenetic Therapy Using Resveratrol and Pterostilbene for Restoring Estrogen Receptor-α (ERα) Expression in ERα-Negative Breast Cancer Cells

    PubMed Central

    Kala, Rishabh; Tollefsbol, Trygve O.

    2016-01-01

    Breast cancer is the second most common cancer and a leading cause of cancer death in women. Specifically, estrogen receptor-α (ERα)-negative breast cancers are clinically more aggressive and normally do not respond to conventional hormone-directed therapies such as tamoxifen. Although epigenetic-based therapies such as 5-aza-2’-deoxycytidine and/or trichostatin A as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, respectively, can regulate the expression of ERα, this can often lead to a number of side effects. Plant-based dietary compounds such as resveratrol and pterostilbene in novel combinatorial therapy provides new avenues to target these side effects and provide similar results with a higher level of safety. Here, we report that combinatorial resveratrol and pterostilbene leads to the reactivation of ERα expression in ERα-negative breast cancer cells in a time-dependent manner. Chromatin immunoprecipitation analysis of the ERα promoter in each cell type revealed an increase in enrichment of acetyl-H3, acetyl-H3lysine9 (H3K9) and acetyl-H4 active chromatin markers in the ERα promoter region after combinatorial treatment. This treatment also resulted in a significant change in HDAC and histone acetyl transferase (HAT) enzyme activity in these cells after 3 days of treatments. The combination resulted in a significant decrease in DNMT enzyme activity and 5-methylcytosine levels in MDA-MB-157 breast cancer cells. Moreover, reactivation of ERα expression by resveratrol combined with pterostilbene was found to sensitize ERα-dependent response to 17β-estradiol (E2)-mediated cellular proliferation and antagonist 4-hydroxytamoxifen (4-OHT)-mediated inhibition of cellular proliferation in ERα-negative breast cancer cells. E2 and 4-OHT further affected the ERα-responsive downstream progesterone receptor (PGR) gene in ERα reactivated MDA-MB-157 cells. Collectively, our findings provide a new and safer way of restoring ER

  13. Involvement of B-cell CLL/lymphoma 2 promoter methylation in cigarette smoke extract-induced emphysema

    PubMed Central

    Zeng, Huihui; Shi, Zhihui; Kong, Xianglong; Chen, Yan; Zhang, Hongliang; Peng, Hong; Luo, Hong

    2016-01-01

    Abnormal apoptotic events play an important role in the pathogenesis of emphysema. The B-cell CLL/lymphoma 2 (Bcl-2) family proteins are essential and critical regulators of apoptosis. We determined whether the anti-apoptotic Bcl-2 play a role in the cigarette smoke extract (CSE)-induced emphysema. Furthermore, given the involvement of epigenetics in chronic obstructive pulmonary disease, we hypothesized that the deregulation of Bcl-2 might be caused by gene methylation. The emphysema in BALB/C mice was established by intraperitoneally injection of CSE. 5-aza-2′-deoxycytidine (AZA; a demethylation reagent) and phosphate-buffered saline were also administered intraperitoneally as CSE. TUNEL assay was used to assess apoptotic index of pulmonary cells. The methylation status of CpG dinucleotides within the Bcl-2 promoter was observed in all groups by bisulfite sequencing PCR. Pulmonary expression of Bcl-2, Bax, and cytochrome C were measured after four weeks of treatment. The apoptotic index of pulmonary cells in CSE injection group was much higher than control ((25.88 ± 7.55)% vs. (6.28 ± 2.96)%). Compared to control mice, decreased expression of Bcl-2 and high methylation of Bcl-2 promoter was observed in CSE injected mice (0.88 ± 0.08 vs. 0.49 ± 0.11, (3.82 ± 1.34)% vs. (35.68 ± 5.99)%, P < 0.01).CSE treatment induced lung cell apoptosis and decreased lung function. AZA treatment increased Bcl-2 expression with Bcl-2 promoter demethylation. AZA also alleviated the lung cell apoptosis and function failure caused by CSE treatment. The decreased expression of anti-apoptotic Bcl-2 might account for the increased apoptosis in CSE induced-emphysema. Apparently, epigenetic alternation played a role in this deregulation of Bcl-2 expression, and it might support the involvement of epigenetic events in the pathogenesis of emphysema. PMID:26924842

  14. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    SciTech Connect

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K.

    2012-08-15

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16{sup INK4a} and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. -- Highlights: ►Epigenetic modulations have been shown to have a role in cancer risk. ►Proanthocyanidins decrease the levels of DNA methylation and histone deacetylation. ►Proanthocyanidins inhibit histone deacetylase activity in skin cancer cells. ►Proanthocyanidins reactivate tumor suppressor genes in skin

  15. Palliative chemotherapy followed by methylation inhibitor in high-risk acute myeloid leukemia: An in vitro and clinical study.

    PubMed

    Ding, Bingjie; Wang, Zhixiang; Jiang, Xuejie; Li, Xiaodong; Wang, Chunli; Zhong, Qingxiu; Jiang, Ling; Dai, Min; Zhang, Y U; Wei, Q I; Meng, Fanyi

    2015-09-01

    Decitabine (5-aza-2'-deoxycytidine; DAC) is a well-tolerated alternative to aggressive chemotherapy for leukemia, which induces differentiation and apoptosis of leukemic cells as a DNA hypomethylating agent. The aim of the present study was to investigate the feasibility of DAC sequentially combined with chemotherapy to reverse drug resistance. HL-60/ADR multidrug-resistant leukemia cells cultured in 96-well plates were pretreated with DAC for 72 h; varying concentrations of aclacinomycin (ACLA) were then added to the wells, cell proliferation was tested using the Cell Counting Kit-8 assay, and DNA methyltransferase 1 (DNMT1) protein expression was detected by western blot analysis. Furthermore, we analyzed the therapeutic efficacy in 7 patients with high-risk acute myeloid leukemia (AML) receiving induction therapy with DAC sequentially combined with cytarabine, ACLA and granulocyte-colony stimulating factor (CAG regimen). The proliferation inhibition rate of HL-60/ADR cells treated with DAC at concentrations of 0.5 and 1.0 µmol/l sequentially combined with ACLA was significantly higher compared with that with ACLA alone (P<0.001 for both). DNMT1 expression was significantly repressed following treatment with 1.0 µmol/l DAC. Of the 11 patients, 8 (72.7%) received induction therapy with DAC sequentially combined with CAG agents and achieved complete remission (CR) after 2 cycles of treatment; however, 3 (27.3%) patients did not achieve remission. Myelosuppression was observed in all 11 patients and pulmonary infections developed in 9 patients (81.8%) during the course of the study. At the last follow-up, 7 of the 8 patients who achieved CR remained in remission. The median follow-up was 6 months (range, 3-18 months). Therefore, pretreatment with DAC may increase the sensitivity of HL-60/ADR cells to ACLA via the epigenetic modulation of demethylation and the sequential administration of DAC and CAG regimen appears to be safe and effective for the treatment of

  16. Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting duct.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2013-10-01

    Aldosterone increases tubular Na(+) absorption largely by increasing α-epithelial Na(+) channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

  17. Mining Gene Expression Signature for the Detection of Pre-Malignant Melanocytes and Early Melanomas with Risk for Metastasis

    PubMed Central

    de Souza, Camila Ferreira; Xander, Patrícia; Monteiro, Ana Carolina; Silva, Amanda Gonçalves dos Santos; da Silva, Débora Castanheira Pereira; Mai, Sabine; Bernardo, Viviane; Lopes, José Daniel; Jasiulionis, Miriam Galvonas

    2012-01-01

    Background Metastatic melanoma is a highly aggressive skin cancer and currently resistant to systemic therapy. Melanomas may involve genetic, epigenetic and metabolic abnormalities. Evidence is emerging that epigenetic changes might play a significant role in tumor cell plasticity and metastatic phenotype of melanoma cells. Principal findings In this study, we developed a systematic approach to identify genes implicated in melanoma progression. To do this, we used the Affymetrix GeneChip Arrays to screen 34,000 mouse transcripts in melan-a melanocytes, 4C pre-malignant melanocytes, 4C11− non-metastatic and 4C11+ metastatic melanoma cell lines. The genome-wide association studies revealed pathways commonly over-represented in the transition from immortalized to pre-malignant stage, and under-represented in the transition from non-metastatic to metastatic stage. Additionally, the treatment of cells with 10 µM 5-aza-2′-deoxycytidine (5AzaCdR) for 48 hours allowed us to identify genes differentially re-expressed at specific stages of melan-a malignant transformation. Treatment of human primary melanocytes with the demethylating agent 5AzaCdR in combination to the histone deacetylase inhibitor Trichostatin A (TSA) revealed changes on melanocyte morphology and gene expression which could be an indicator of epigenetic flexibility in normal melanocytes. Moreover, changes on gene expression recognized by affecting the melanocyte biology (NDRG2 and VDR), phenotype of metastatic melanoma cells (HSPB1 and SERPINE1) and response to cancer therapy (CTCF, NSD1 and SRC) were found when Mel-2 and/or Mel-3-derived patient metastases were exposed to 5AzaCdR plus TSA treatment. Hierarchical clustering and network analyses in a panel of five patient-derived metastatic melanoma cells showed gene interactions that have never been described in melanomas. Significance Despite the heterogeneity observed in melanomas, this study demonstrates the utility of our murine melanoma

  18. Functional role and tobacco smoking effects on methylation of CYP1A1 gene in prostate cancer

    PubMed Central

    Kato, Taku; Hashimoto, Yutaka; Yamamura, Soichiro; Fukuhara, Shinichiro; Wong, Darryn K.; Shiina, Marisa; Imai-Sumida, Mitsuho; Majid, Shahana; Saini, Sharanjot; Shiina, Hiroaki; Nakajima, Koichi; Deng, Guoren; Dahiya, Rajvir; Tanaka, Yuichiro

    2016-01-01

    Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2′-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment. PMID:27203547

  19. Valproic Acid Increases CD133 Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma

    PubMed Central

    Khalil, Mohamed Ashraf; Hraběta, Jan; Groh, Tomáš; Procházka, Pavel; Doktorová, Helena; Eckschlager, Tomáš

    2016-01-01

    Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through its action as a histone deacetylase inhibitor. CD133 is considered to be a cancer stem cell marker in several tumors including neuroblastoma. CD133 transcription is strictly regulated by epigenetic modifications. We evaluated the epigenetic effects of treatment with 1mM VPA and its influence on the expression of CD133 in four human neuroblastoma cell lines. Chemoresistance and cell cycle of CD133+ and CD133− populations were examined by flow cytometry. We performed bisulfite conversion followed by methylation-sensitive high resolution melting analysis to assess the methylation status of CD133 promoters P1 and P3. Our results revealed that VPA induced CD133 expression that was associated with increased acetylation of histones H3 and H4. On treatment with VPA and cytostatics, CD133+ cells were mainly detected in the S and G2/M phases of the cell cycle and they showed less activated caspase-3 compared to CD133− cells. UKF-NB-3 neuroblastoma cells which express CD133 displayed higher colony and neurosphere formation capacities when treated with VPA, unlike IMR-32 which lacks for CD133 protein. Induction of CD133 in UKF-NB-3 was associated with increased expression of phosphorylated Akt and pluripotency transcription factors Nanog, Oct-4 and Sox2. VPA did not induce CD133 expression in cell lines with methylated P1 and P3 promoters, where the CD133 protein was not detected. Applying the demethylating agent 5-aza-2’-deoxycytidine to the cell lines with methylated promoters resulted in CD133 re-expression that was associated with a drop in P1 and P3 methylation level. In conclusion, CD133 expression in neuroblastoma can be regulated by histone acetylation and/or methylation of its CpG promoters. VPA can induce CD133+ cells which display high proliferation potential and low sensitivity to cytostatics in neuroblastoma. These results give new insight into the possible

  20. DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

    PubMed

    Venza, Mario; Visalli, Maria; Catalano, Teresa; Biondo, Carmelo; Beninati, Concetta; Teti, Diana; Venza, Isabella

    2016-04-01

    E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing

  1. Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK.

    PubMed

    Pan, Feng-Ping; Zhou, Hong-Kun; Bu, He-Qi; Chen, Zi-Qiang; Zhang, Hao; Xu, Lu-Ping; Tang, Jian; Yu, Qing-Jiang; Chu, Yong-Quan; Pan, Jie; Fei, Yong; Lin, Sheng-Zhang; Liu, Dian-Lei; Chen, Liang

    2016-04-01

    5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor

  2. Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids.

    PubMed

    Kato, Norihiro; Yamamoto, Hiroyuki; Adachi, Yasushi; Ohashi, Hirokazu; Taniguchi, Hiroaki; Suzuki, Hiromu; Nakazawa, Mayumi; Kaneto, Hiroyuki; Sasaki, Shigeru; Imai, Kohzoh; Shinomura, Yasuhisa

    2013-03-21

    fluids was most useful for the detection of pancreatic and pancreatobiliary cancers, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancers. Treatment with a demethylating agent, 5-AZA-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines. Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.

  3. Neonatal isolation decreases cued fear conditioning and frontal cortical histone 3 lysine 9 methylation in adult female rats.

    PubMed

    Kao, Gour-Shenq; Cheng, Ling-Yi; Chen, Li-Hsien; Tzeng, Wen-Yu; Cherng, Chienfang G; Su, Chien-Chou; Wang, Ching-Yi; Yu, Lung

    2012-12-15

    Early life stress is thought to enhance adult susceptibility to stress and stress-related mood disorders. In this study, fear-potentiated startle was used to model the acquisition of a traumatic event-related memory in female rats experiencing early life stress. Daily 1-hr maternal and sibling separation throughout day 2-9 postpartum (D2-9 PP) caused a decrease in the fear-potentiated startle, but not acoustic startle baseline, in adult female rats. The separation procedure did not affect corticosterone secretion but produced an increase in serum estradiol concentration. Moreover, the separation procedure did not affect histone 3 lysine 9 (H3K9) acetylation but decreased H3K9 mono- and tri-methylation in frontal cortices. Treatment with 5-aza-2'-deoxycytidine (AZA) (5mg/kg at alternative days from D2PP to D9PP or 10mg/kg at D5PP and D9PP), a DNA methylation inhibitor, did not affect the separation-decreased fear-potentiated startle. Treatment with valproic acid (VPA), a histone deacetylase inhibitor, at 3 dosing regimens (300mg/kg at D2-9PP; 100mg/kg at D2-4PP, 200mg/kg at D5-7PP, 300mg/kg at D8-9PP; 100mg/kg at D2-5PP, 200mg/kg at D6-9PP) prior to daily separation reversed such a decrease in fear-potentiated startle. The lowest effective VPA dosing regimen used (100mg/kg at D2-5PP, 200mg/kg at D6-9PP) reversed the separation-decreased H3K9 mono- and tri-methylation in frontal cortices. Eight-day VPA (300mg/kg/day) and AZA (5mg/kg/day) administrations starting at D28PP were ineffective in altering the separation-decreased fear-potentiated startle. We, hereby, suggest that decreased frontal cortical H3K9 mono- and tri-methylation may be involved in early life separation-decreased fear memory of adult rats.

  4. Identification of Predictive DNA Methylation Biomarkers for Chemotherapy Response in Colorectal Cancer

    PubMed Central

    Baharudin, Rashidah; Ab Mutalib, Nurul-Syakima; Othman, Sri N.; Sagap, Ismail; Rose, Isa M.; Mohd Mokhtar, Norfilza; Jamal, Rahman

    2017-01-01

    Resistance to 5-Fluorouracil (5-FU) is a major obstacle to the successful treatment of colorectal cancer (CRC) and posed an increased risk of recurrence. DNA methylation has been suggested as one of the underlying mechanisms for recurrent disease and its contribution to the development of drug resistance remains to be clarified. This study aimed to determine the methylation phenotype in CRC for identification of predictive markers for chemotherapy response. We performed DNA methylation profiling on 43 non-recurrent and five recurrent CRC patients using the Illumina Infinium HumanMethylation450 Beadchip assay. In addition, CRC cells with different genetic backgrounds, response to 5-FU and global methylation levels (HT29 and SW48) were treated with 5-FU and DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC). The singular and combined effects of these two drug classes on cell viability and global methylation profiles were investigated. Our genome-wide methylation study on the clinical specimens showed that recurrent CRCs exhibited higher methylation levels compared to non-recurrent CRCs. We identified 4787 significantly differentially methylated genes (P < 0.05); 3112 genes were hyper- while 1675 genes were hypomethylated in the recurrent group compared to the non-recurrent. Fifty eight and 47 of the significantly hypermethylated and hypomethylated genes have an absolute recurrent/non-recurrent methylation difference of ≥20%. Most of the hypermethylated genes were involved in the MAPK signaling pathway which is a key regulator for apoptosis while the hypomethylated genes were involved in the PI3K-AKT signaling pathway and proliferation process. We also demonstrate that 5-azadC treatment enhanced response to 5-FU which resulted in significant growth inhibition compared to 5-FU alone in hypermethylated cell lines SW48. In conclusion, we found the evidence of five potentially biologically important genes in recurrent CRCs that could possibly serve as a new

  5. Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK

    PubMed Central

    PAN, FENG-PING; ZHOU, HONG-KUN; BU, HE-QI; CHEN, ZI-QIANG; ZHANG, HAO; XU, LU-PING; TANG, JIAN; YU, QING-JIANG; CHU, YONG-QUAN; PAN, JIE; FEI, YONG; LIN, SHENG-ZHANG; LIU, DIAN-LEI; CHEN, LIANG

    2016-01-01

    5-Aza-2′-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of meth-yltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of

  6. 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

  7. Epigenetic inactivation of EFEMP1 is associated with tumor suppressive function in endometrial carcinoma.

    PubMed

    Yang, Tingting; Qiu, Haifeng; Bao, Wei; Li, Bilan; Lu, Cong; Du, Guiqiang; Luo, Xin; Wang, Lihua; Wan, Xiaoping

    2013-01-01

    EFEMP1, the epidermal growth factor-containing fibulin-like extracellular matrix protein 1, functions as an oncogene or a tumor suppressor depending on the cancer types. In this study, we aim to determine whether EFEMP1 affects the tumorigenesis and progression of endometrial carcinoma. The expression of EFEMP1 was investigated using immunohistochemistry in a panel of normal endometrium (n = 40), atypical hyperplasia (n = 10) and endometrial carcinoma tissues (n = 84). Methylation status of the EFEMP1 promoter was detected by methylation-specific PCR (MSP) and bisulphite genomic sequencing. Up- or down-regulation of EFEMP1 were achieved by stable or transient transfection with pCMV6/GFP/Neo-EFEMP1 or pGPU6/GFP/Neo-shEFEMP1 respectively. Effects of EFEMP1 on tumor proliferation, invasion and migration were evaluated by MTT, plate colony formation, Transwell and wound healing assay. The nude mouse tumor xenograft assay was used to investigate function of EFEMP1 in vivo. Compared with normal endometrium (32/40) and atypical hyperplasia (7/10), EFEMP1 expression was much lower in endometrial carcinoma tissues (16/84) (P<0.001 and P = 0.02). EFEMP1 promoter was hypermethylated in endometrial carcinoma tissues (67%) as compared to normal tissue (10%) and down-regulation of EFEMP1 was associated with promoter hypermethylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) altered EFEMP1 methylation status, and restored EFEMP1 expression. Moreover, EFEMP1 decreased secretion of MMPs and inhibited tumor cell proliferation, metastasis and invasion in vitro and suppressed tumorigenesis in nude mice. Besides, EFEMP1 increased expression of E-cadherin and suppressed expression of vimentin in endometrial carcinoma. EFEMP1 is a new candidate tumor suppressor gene in endometrial carcinoma, and is frequently silenced by promoter hypermethylation. It could inhibit tumor growth and invasion both in vitro and in vivo. Our findings propose that targeting

  8. Epigenetic Inactivation of EFEMP1 Is Associated with Tumor Suppressive Function in Endometrial Carcinoma

    PubMed Central

    Yang, Tingting; Qiu, Haifeng; Bao, Wei; Li, Bilan; Lu, Cong; Du, Guiqiang; Luo, Xin; Wang, Lihua; Wan, Xiaoping

    2013-01-01

    Objective EFEMP1, the epidermal growth factor–containing fibulin-like extracellular matrix protein 1, functions as an oncogene or a tumor suppressor depending on the cancer types. In this study, we aim to determine whether EFEMP1 affects the tumorigenesis and progression of endometrial carcinoma. Methods The expression of EFEMP1 was investigated using immunohistochemistry in a panel of normal endometrium (n = 40), atypical hyperplasia (n = 10) and endometrial carcinoma tissues (n = 84). Methylation status of the EFEMP1 promoter was detected by methylation-specific PCR (MSP) and bisulphite genomic sequencing. Up- or down-regulation of EFEMP1 were achieved by stable or transient transfection with pCMV6/GFP/Neo-EFEMP1 or pGPU6/GFP/Neo-shEFEMP1 respectively. Effects of EFEMP1 on tumor proliferation, invasion and migration were evaluated by MTT, plate colony formation, Transwell and wound healing assay. The nude mouse tumor xenograft assay was used to investigate function of EFEMP1 in vivo. Results Compared with normal endometrium (32/40) and atypical hyperplasia (7/10), EFEMP1 expression was much lower in endometrial carcinoma tissues (16/84) (P<0.001 and P = 0.02). EFEMP1 promoter was hypermethylated in endometrial carcinoma tissues (67%) as compared to normal tissue (10%) and down-regulation of EFEMP1 was associated with promoter hypermethylation. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) altered EFEMP1 methylation status, and restored EFEMP1 expression. Moreover, EFEMP1 decreased secretion of MMPs and inhibited tumor cell proliferation, metastasis and invasion in vitro and suppressed tumorigenesis in nude mice. Besides, EFEMP1 increased expression of E-cadherin and suppressed expression of vimentin in endometrial carcinoma. Conclusion EFEMP1 is a new candidate tumor suppressor gene in endometrial carcinoma, and is frequently silenced by promoter hypermethylation. It could inhibit tumor growth and invasion both

  9. Multiple-to-Multiple Relationships between MicroRNAs and Target Genes in Gastric Cancer

    PubMed Central

    Hashimoto, Yutaka; Akiyama, Yoshimitsu; Yuasa, Yasuhito

    2013-01-01

    MicroRNAs (miRNAs) act as transcriptional regulators and play pivotal roles in carcinogenesis. According to miRNA target databases, one miRNA may regulate many genes as its targets, while one gene may be targeted by many miRNAs. These findings indicate that relationships between miRNAs and their targets may not be one-to-one. However, many reports have described only a one-to-one, one-to-multiple or multiple-to-one relationship between miRNA and its target gene in human cancers. Thus, it is necessary to determine whether or not a combination of some miRNAs would regulate multiple targets and be involved in carcinogenesis. To find some groups of miRNAs that may synergistically regulate their targets in human gastric cancer (GC), we re-analyzed our previous miRNA expression array data and found that 50 miRNAs were up-regulated on treatment with 5-aza-2'-deoxycytidine in a GC cell line. The “TargetScan” miRNA target database predicted that some of these miRNAs have common target genes. We also referred to the GEO database for expression of these common target genes in human GCs, which might be related to gastric carcinogenesis. In this study, we analyzed two miRNA combinations, miR-224 and -452, and miR-181c and -340. Over-expression of both miRNA combinations dramatically down-regulated their target genes, DPYSL2 and KRAS, and KRAS and MECP2, respectively. These miRNA combinations synergistically decreased cell proliferation upon transfection. Furthermore, we revealed that these miRNAs were down-regulated through promoter hypermethylation in GC cells. Thus, it is likely that the relationships between miRNAs and their targets are not one-to-one but multiple-to-multiple in GCs, and that these complex relationships may be related to gastric carcinogenesis. PMID:23667495

  10. Amyloid Beta-Mediated Hypomethylation of Heme Oxygenase 1 Correlates with Cognitive Impairment in Alzheimer’s Disease

    PubMed Central

    Sung, Hye Youn; Choi, Byung-Ok; Jeong, Jee Hyang; Kong, Kyoung Ae; Hwang, Jinha; Ahn, Jung-Hyuck

    2016-01-01

    To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer’s disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located −374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2′-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aβ-induced aberrant hypomethylation of HMOX1 at −374 promoter CpG site was correlated with increased HMOX1expression. In addition to neuroglioma cells, we also found Aβ-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at −374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the −374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and

  11. Decitabine has a biphasic effect on natural killer cell viability, phenotype, and function under proliferative conditions.

    PubMed

    Kopp, Lisa M; Ray, Anish; Denman, Cecele J; Senyukov, Vladimir S; Somanchi, Srinivas S; Zhu, Shiguo; Lee, Dean A

    2013-07-01

    DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).(4) The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy. The cytidine analog 5-aza-2'-deoxycytidine (decitabine, DAC) has both a hypomethylating effect at low doses when incorporated into DNA and a cytotoxic effect at higher doses as a result of interfering with translation when incorporated into RNA. Thus, decitabine has been used at higher doses for its direct anti-leukemic effect, and is being tested at low doses for its ability to correct the malignant gene expression phenotype. A known benefit of hypomethylating agents is their ability to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct effect of hypomethylating agents on NK cell phenotype, proliferation, survival, or function. We recently described a method for inducing robust proliferation of NK cells, enabling us to study the hypomethylating effects of decitabine. To distinguish direct toxicity of the decitabine from its hypomethylating effect, and promote hypomethylation during proliferation, decitabine was added to human peripheral blood NK cells at concentrations from 0.02 to 5μM under either static or proliferation-inducing culture conditions. After 5 days, NK cells were assessed for viability, proliferation, cytotoxicity, expression of major activating and inhibitory receptors, and global DNA methylation. Increasing concentrations of decitabine not only causes increased expression of KIR and the activating

  12. Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers.

    PubMed

    Dallol, Ashraf; Forgacs, Eva; Martinez, Alonso; Sekido, Yoshitaka; Walker, Rosemary; Kishida, Takeshi; Rabbitts, Pamela; Maher, Eamonn R; Minna, John D; Latif, Farida

    2002-05-02

    The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for

  13. Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

    PubMed

    Tsang, Daisy P F; Wu, William K K; Kang, Wei; Lee, Ying-Ying; Wu, Feng; Yu, Zhuo; Xiong, Lei; Chan, Anthony W; Tong, Joanna H; Yang, Weiqin; Li, May S M; Lau, Suki S; Li, Xiangchun; Lee, Sau-Dan; Yang, Yihua; Lai, Paul B S; Yu, Dae-Yeul; Xu, Gang; Lo, Kwok-Wai; Chan, Matthew T V; Wang, Huating; Lee, Tin L; Yu, Jun; Wong, Nathalie; Yip, Kevin Y; To, Ka-Fai; Cheng, Alfred S L

    2016-04-01

    Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

  14. MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.

    PubMed

    Iwaki, Jun; Kikuchi, Kunio; Mizuguchi, Yoshiaki; Kawahigashi, Yutaka; Yoshida, Hiroshi; Uchida, Eiji; Takizawa, Toshihiro

    2013-01-01

    MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376

  15. MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

    PubMed Central

    Mizuguchi, Yoshiaki; Kawahigashi, Yutaka; Yoshida, Hiroshi; Uchida, Eiji; Takizawa, Toshihiro

    2013-01-01

    MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that mi

  16. Inhibition of DNA Methylation and Methyl-CpG-Binding Protein 2 Suppresses RPE Transdifferentiation: Relevance to Proliferative Vitreoretinopathy

    PubMed Central

    He, Shikun; Barron, Ernesto; Ishikawa, Keijiro; Nazari Khanamiri, Hossein; Spee, Chris; Zhou, Peng; Kase, Satoru; Wang, Zhuoshi; Dustin, Laurie Diane; Hinton, David R.

    2015-01-01

    Purpose The purpose of this study was to evaluate expression of methyl-CpG-binding protein 2 (MeCP2) in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR) and to investigate effects of inhibition of MeCP2 and DNA methylation on transforming growth factor (TGF)-β–induced retinal pigment epithelial (RPE) cell transdifferentiation. Methods Expression of MeCP2 and its colocalization with cytokeratin and α-smooth muscle actin (α-SMA) in surgically excised PVR membranes was studied using immunohistochemistry. The effects of 5-AZA-2′-deoxycytidine (5-AZA-dC) on human RPE cell migration and viability were evaluated using a modified Boyden chamber assay and the colorimetric 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Expression of RASAL1 mRNA and its promoter region methylation were evaluated by real-time PCR and methylation-specific PCR. Effects of 5-AZA-dC on expression of α-SMA, fibronectin (FN), and TGF-β receptor 2 (TGF-β R2) and Smad2/3 phosphorylation were analyzed by Western blotting. Effect of short interfering RNA (siRNA) knock-down of MeCP2 on expression of α-SMA and FN induced by TGFβ was determined. Results MeCP2 was abundantly expressed in cells within PVR membranes where it was double labeled with cells positive for cytokeratin and α-SMA. 5-AZA-dC inhibited expression of MeCP2 and suppressed RASAL1 gene methylation while increasing expression of the RASAL1 gene. Treatment with 5-AZA-dC significantly suppressed the expression of α-SMA, FN, TGF-β R2 and phosphorylation of Smad2/3 and inhibited RPE cell migration. TGF-β induced expression of α-SMA, and FN was suppressed by knock-down of MeCP2. Conclusions MeCP2 and DNA methylation regulate RPE transdifferentiation and may be involved in the pathogenesis of PVR. PMID:26305530

  17. Curcumin modulates DNA methylation in colorectal cancer cells.

    PubMed

    Link, Alexander; Balaguer, Francesc; Shen, Yan; Lozano, Juan Jose; Leung, Hon-Chiu E; Boland, C Richard; Goel, Ajay

    2013-01-01

    Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza

  18. CHST11 gene expression and DNA methylation in breast cancer

    PubMed Central

    HERMAN, DAMIR; LEAKEY, TATIANA I.; BEHRENS, ALICE; YAO-BORENGASSER, AIWEI; COONEY, CRAIG A.; JOUSHEGHANY, FARIBA; PHANAVANH, BOUNLEUT; SIEGEL, ERIC R.; SAFAR, A. MAZIN; KOROURIAN, SOHEILA; KIEBER-EMMONS, THOMAS; MONZAVI-KARBASSI, BEHJATOLAH

    2015-01-01

    Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2′-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the

  19. DNA methyltransferases as targets for cancer therapy.

    PubMed

    Ghoshal, Kalpana; Bai, Shoumei

    2007-06-01

    Methylation of DNA at 5-position of cytosine, catalyzed by DNA methyltransferases, is the predominant epigenetic modification in mammals. Aberrations in methylation play a causal role in a variety of diseases, including cancer. Recent studies have established that like mutation, methylation-mediated gene silencing often leads to tumorigenesis. Paradoxically, genome-wide DNA hypomethylation may also play a causal role in carcinogenesis by inducing chromosomal instability and spurious gene expression. Since methylation does not alter DNA base sequence, much attention has been focused recently on developing small molecule inhibitors of DNA methyltransferases that can potentially be used as anticancer agents. Vidaza (5-azacytidine), marketed by Pharmion (Boulder, CO, USA), was the first DNA methyltransferase inhibitor approved by the U.S. Food and Drug Administration (FDA) for chemotherapy against myelodysplastic syndrome (MDS), a heterogeneous bone marrow disorder. Recently MGI Pharma Inc. (Bloomington, MN, USA) got FDA approval to market Dacogen (5-aza-2'-deoxycytidine, or decitabine) for treating MDS patients. These drugs were used earlier against certain anemias to induce expression of fetal globin genes. Interest in clinical trials of these drugs as anticancer agents has been renewed only recently because of reversal of methylation-mediated silencing of critical genes in cancer. Clinical trials have shown that both drugs have therapeutic potential against leukemia such as MDS, acute myeloid leukemia, chronic myelogenous leukemia and chronic myelomonocytic leukemia. In contrast, their effectiveness with solid tumors appears to be less promising, which challenges researchers to develop inhibitors with more efficacy and less toxicity. The major hindrance of their usage as anticancer agents is their instability in vivo as well as the toxicity secondary to their excessive incorporation into DNA, which causes cell cycle arrest. Gene expression profiling in cancer cells

  20. Epigenetic silencing of glutaminase 2 in human liver and colon cancers

    PubMed Central

    2013-01-01

    Background Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers. Methods mRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test. Results First, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest. Conclusions We provide evidence showing that epigenetic silencing of Gls2 via promoter

  1. The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24).

    PubMed

    Varol, Nuray; Konac, Ece; Onen, Ilke Hacer; Gurocak, Serhat; Alp, Ebru; Yilmaz, Akin; Menevse, Sevda; Sozen, Sinan

    2014-07-01

    The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both β-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 μM), DAC (10 μM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3β, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of β-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/β-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3β mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/β-catenin pathway and reduced

  2. 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

  3. Expression of the candidate tumor suppressor gene hSRBC is frequently lost in primary lung cancers with and without DNA methylation.

    PubMed

    Zöchbauer-Müller, Sabine; Fong, Kwun M; Geradts, Joseph; Xu, Xie; Seidl, Sonja; End-Pfützenreuter, Adelheid; Lang, György; Heller, Gerwin; Zielinski, Christoph C; Gazdar, Adi F; Minna, John D

    2005-09-15

    Recently, the human SRBC (hSRBC) gene, a candidate tumor suppressor gene (TSG), has been mapped to the chromosomal region 11p 15.5--p15.4 where frequent allele loss has been described in lung cancer. Aberrant methylation (referred to as methylation) of the promoter region of TSGs has been identified as an important mechanism for gene silencing. Loss of hSRBC protein expression occurs frequently in lung cancer cell lines and sodium bisulfite sequencing of the promoter region of hSRBC in several lung cancer cell lines suggested that methylation plays an important role in inactivating hSRBC. To determine the methylation status of hSRBC in a large collection of primary lung cancer samples, corresponding nonmalignant lung tissues and lung cancer cell lines (N=52), we designed primers for a methylation-specific PCR assay. Methylation was detected in 41% of primary non-small-cell lung cancers (NSCLC) (N=107) and in 80% of primary small-cell lung cancers (SCLC) (N=5), but was seen only in 4% of corresponding nonmalignant lung tissues (N=103). In all, 79% of lung cancer cell lines were methylated and the frequency of hSRBC methylation was significantly higher in SCLC (100%) than in NSCLC (58%) cell lines. Normal hSRBC protein expression was detected in only 18% of primary NSCLCs (N=93) by immunostaining and a significant association between loss of protein expression and methylation was found. hSRBC re-expression was observed after treatment of lung cancer cells with the demethylating agent 5-aza-2'-deoxycytidine. In addition, 45% of the 76 hSRBC immunostaining-negative NSCLCs did not have hSRBC promoter methylation, indicating that other mechanisms of hSRBC expression silencing also exist. Both hSRBC immunostaining and methylation results did not correlate with clinicopathological characteristics of these patients. Our findings suggest that hSRBC is a candidate TSG involved in lung cancer pathogenesis, where expression is frequently inactivated by methylation and other

  4. Amyloid Beta-Mediated Hypomethylation of Heme Oxygenase 1 Correlates with Cognitive Impairment in Alzheimer's Disease.

    PubMed

    Sung, Hye Youn; Choi, Byung-Ok; Jeong, Jee Hyang; Kong, Kyoung Ae; Hwang, Jinha; Ahn, Jung-Hyuck

    2016-01-01

    To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer's disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located -374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2'-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aβ-induced aberrant hypomethylation of HMOX1 at -374 promoter CpG site was correlated with increased HMOX1 expression. In addition to neuroglioma cells, we also found Aβ-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at -374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the -374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and control

  5. Antitumor activity of epigenetic immunomodulation combined with CTLA-4 blockade in syngeneic mouse models

    PubMed Central

    Covre, A; Coral, S; Nicolay, H; Parisi, G; Fazio, C; Colizzi, F; Fratta, E; Di Giacomo, A M; Sigalotti, L; Natali, P G; Maio, M

    2015-01-01

    The multifaceted immunomodulatory activity of DNA hypomethylating agents improves immunogenicity and immune recognition of neoplastic cells; thus, we predicted they could be utilized to design new immunotherapeutic combinations in cancer. Testing this hypothesis, the antitumor efficacy of the DNA hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA-CdR) combined with the anti-CTLA-4 monoclonal antibody (mAb) 9H10 in syngeneic transplantable murine models was investigated. Murine mammary carcinoma TS/A or mesothelioma AB1 cells were injected in BALB/c, athymic nude, and SCID/Beige mice that were treated with 5-AZA-CdR, mAb 9H10, or their combination. Tumor volumes were captured at different time-points; molecular and immunohistochemical assays investigated changes in neoplastic and normal tissues. A significant antitumor effect of 5-AZA-CdR combined with mAb 9H10 was found: compared to controls, a 77% (p < 0.01), 54% (p < 0.01) and 33% (p = 0.2) decrease in TS/A tumor growth was induced by 5-AZA-CdR combined with mAb 9H10, 5-AZA-CdR or mAb 9H10, respectively. These antitumor activities were confirmed utilizing the AB1 model. 5-AZA-CdR-based regimens induced a promoter-demethylation-sustained tumor expression of cancer testis antigens. MHC class I expression was up-regulated by 5-AZA-CdR. Antitumor efficacy of 5-AZA-CdR in athymic nude and SCID/Beige mice was not increased by mAb 9H10. In BALB/c mice, combined treatment induced the highest tumor infiltration by CD3+ lymphocytes, which included both CD8+ and CD4+ T cells; no such infiltrates were observed in normal tissues. This significant immune-related antitumor activity of 5-AZA-CdR combined with CTLA-4 blockade, demonstrated in highly aggressive mouse tumor models, provides a strong scientific rationale to implement epigenetically-based immunotherapies in cancer patients. PMID:26405573

  6. Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer.

    PubMed

    To, Sarah Q; Takagi, Kiyoshi; Miki, Yasuhiro; Suzuki, Koyu; Abe, Eriko; Yang, Yang; Sasano, Hironobu; Simpson, Evan R; Knower, Kevin C; Clyne, Colin D

    2012-11-01

    The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a

  7. Prognostic value of CpG island hypermethylation at PTGS2, RAR-beta, EDNRB, and other gene loci in patients undergoing radical prostatectomy.

    PubMed

    Bastian, Patrick J; Ellinger, Jörg; Heukamp, Lukas C; Kahl, Philip; Müller, Stefan C; von Rücker, Alexander

    2007-03-01

    To evaluate CpG island hypermethylation in a set of candidate genes in prostate cancer (pCA) and its relationship to clinicopathologic parameters and a nomogram predicting prostate-specific antigen (PSA) recurrence after radical prostatectomy. Tissues of 78 prostate carcinomas, 32 benign prostate hyperplasias (BPHs), and prostate cell lines (LNCaP, DU145, PC3, BPH-1) were examined with MethyLight polymerase chain reaction at 13 gene loci (APC, CDC6, CTNNB1, E-Cadherin, EDNRB, FGFR2, GSTP1, NAB2, PKCmu, PTGS2, RAR-beta, RASL11A, WWOX). APC, RAR-beta, PTGS2, GSTP1, EDNRB, and CTNNB1 (83%, 71%, 65%, 33%, 14%, 9%, respectively) were methylated in pCA but rarely or not methylated in BPH. NAB2 and CDC6 were hypermethylated frequently in pCA (92%, 67%, respectively) and in BPH (91%, 59%, respectively). FGFR2, WWOX, E-Cadherin, PKCmu, and RASLL1A did not display noteworthy methylation in pCA (0-1%) or in BPH. CpG island hypermethylation at APC, retinoic acid receptor beta (RAR-beta), and PTGS2 discriminated with a sensitivity of 65-83% and a specificity of 97-100% between BPH and pCA. The combination of various genes increased the diagnostic expressiveness. PTGS2 hypermethylation correlated with seminal vesicle infiltration (p=0.047), capsular penetration (p=0.004), and pT stage (p=0.014). RAR-beta methylation was accompanied by a higher cumulative Gleason score (p=0.042). The probability of PSA-free-survival calculated with a Kattan nomogram correlated inversely with CpG island hypermethylation at EDNRB, RAR-beta, and PTGS2. All prostate cancer cell lines displayed a varying degree of demethylation after 5-aza-2'deoxycytidine treatment. CpG island hypermethylation at various gene loci is frequent in prostate cancer and can distinguish between neoplastic and noncancerous tissue. Furthermore, hypermethylation at PTGS2, RAR-beta, and EDNRB inversely correlated with PSA-free-survival according to a Kattan nomogram and has potential prognostic value.

  8. A new tumor suppressor lncRNA RP11-190D6.2 inhibits the proliferation, migration, and invasion of epithelial ovarian cancer cells

    PubMed Central

    Tong, Wenxian; Yang, Liu; Yu, Qiang; Yao, Jie; He, Anbing

    2017-01-01

    At present, a large number of long noncoding RNAs (lncRNAs) from the human genome have been discovered. Meanwhile, emerging evidence has indicated that lncRNAs could play a critical role in the regulation of cellular processes such as cancer progression and metastasis. However, the functions of some new lncRNAs in the complex transcriptional process are mostly unknown at present. Existing studies suggest that loss of WW domain-containing oxidoreductase (WWOX) expression is linked with poor prognosis in numerous cancers, including epithelial ovarian cancer (EOC). However, the functional role of its antisense transcript RP11-190D6.2 is not clear to date. In this study, WWOX antisense transcript RP11-190D6.2 was analyzed specifically in EOC cells using real-time polymerase chain reaction and gain-/loss-of-function studies. We found that RP11-190D6.2 expression was positively correlated with WWOX expression. The RP11-190D6.2 expression was markedly downregulated in tumor tissues compared with normal tissues, but the RP11-190D6.2 expression was significantly downregu-lated in four EOC cell lines compared with human ovarian surface epithelial cell line. RP11-190D6.2 overexpression resulted in the increase of WWOX expression, whereas its knockdown led to the decrease of WWOX expression. We also found that RP11-190D6.2 was restored by 5-aza-2′-deoxycytidine treatment in EOC. In addition, the RP11-190D6.2 overexpression and knockdown experiments revealed that RP11-190D6.2 overexpression inhibited proliferation, migration, and invasion abilities in HO8910-PM cells, whereas RP11-190D6.2 knockdown in HEY-A8 cells had the opposite effect. The analyses in EOC implicate that RP11-190D6.2 may play a pivotal role in the regulation of tumor metastasis, suggesting that RP11-190D6.2 may serve as a potential biomarker and therapeutic target for EOC. PMID:28280357

  9. CHST11 gene expression and DNA methylation in breast cancer.

    PubMed

    Herman, Damir; Leakey, Tatiana I; Behrens, Alice; Yao-Borengasser, Aiwei; Cooney, Craig A; Jousheghany, Fariba; Phanavanh, Bounleut; Siegel, Eric R; Safar, A Mazin; Korourian, Soheila; Kieber-Emmons, Thomas; Monzavi-Karbassi, Behjatolah

    2015-03-01

    Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2'-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the

  10. Activin signaling in microsatellite stable colon cancers is disrupted by a combination of genetic and epigenetic mechanisms.

    PubMed

    Jung, Barbara; Gomez, Jessica; Chau, Eddy; Cabral, Jennifer; Lee, Jeffrey K; Anselm, Aimee; Slowik, Przemyslaw; Ream-Robinson, Deena; Messer, Karen; Sporn, Judith; Shin, Sung K; Boland, C Richard; Goel, Ajay; Carethers, John M

    2009-12-14

    Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers. Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2'-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria. Of 51 MSS colon tumors, 7 (14%) lost ACVR2, 2 (4%) ACVR1, and 5 (10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05). Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.

  11. Activin Signaling in Microsatellite Stable Colon Cancers Is Disrupted by a Combination of Genetic and Epigenetic Mechanisms

    PubMed Central

    Jung, Barbara; Gomez, Jessica; Chau, Eddy; Cabral, Jennifer; Lee, Jeffrey K.; Anselm, Aimee; Slowik, Przemyslaw; Ream-Robinson, Deena; Messer, Karen; Sporn, Judith; Shin, Sung K.; Boland, C. Richard; Goel, Ajay; Carethers, John M.

    2009-01-01

    Background Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers. Methods Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria. Results Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05). Conclusions Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer. PMID:20011542

  12. Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell lines.

    PubMed

    Lambrot, R; Kimmins, S

    2011-04-01

    DNA and histone methylation are epigenetic modifications functioning in transcriptional control and have been implicated in the deregulation of gene expression in cancer. As a first step to determine if histone methylation could be involved in testis cancer pathogenesis, we performed immunofluorescent localization of histone H3 methylation at lysine 4 (H3-K4; gene activating) and lysine 9 (H3-K9; gene silencing) in healthy testis tissue and in samples of non-seminoma germ-cell tumours. In healthy testis, the distribution of histone H3 methylation was dependent on the development