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Sample records for abcg2 promoter demethylation

  1. Chemotherapeutic Drug-Induced ABCG2 Promoter Demethylation as a Novel Mechanism of Acquired Multidrug Resistance

    Eran E. Bram

    2009-12-01

    Full Text Available ABCG2 is an efflux transporter conferring multidrug resistance (MDR on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

  2. Generation of an ABCG2GFPn-puro transgenic line - A tool to study ABCG2 expression in mice

    The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.

  3. ABCG2 Localizes to the Nucleus and Modulates CDH1 Expression in Lung Cancer Cells

    Shu-Ching Liang

    2015-03-01

    Full Text Available Breast cancer resistance protein [BCRP/ATP-binding cassette subfamily G member 2 (ABCG2] is a member of the ATP-binding cassette transporter family. The presence of ABCG2 on the plasma membrane in many kinds of human cancer cells contributes to multidrug resistance during chemotherapy, and it has been used as the side population marker for identifying cancer stem cells in lung cancers. We report here that, in addition to the membranous form, ABCG2 proteins are also found inside the nucleus, where they bind to the E-box of CDH1 (E-cadherin promoter and regulate transcription of this gene. Increased expression of ABCG2 causes an increase of E-cadherin and attenuates cell migration, whereas knockdown of ABCG2 downregulates E-cadherin and enhances cell motility. In mice, xenografted A549 cells that have less ABCG2 are more likely to metastasize from the subcutaneous inoculation site to the internal organs. However, for the cancer cells that have already entered the blood circulation, an increased level of ABCG2, and correspondingly increased E-cadherin, may facilitate circulating cancer cells to colonize at a distant site and form a metastatic tumor. We propose a novel role for nuclear ABCG2 that functions as a transcription regulator and participates in modulation of cancer metastasis.

  4. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    Oberstadt, Moritz C.; Bien-Möller, Sandra; Weitmann, Kerstin; Herzog, Susann; Hentschel, Katharina; Rimmbach, Christian; Vogelgesang, Silke; Balz, Ellen; Fink, Matthias; Michael, Heike; Zeden, Jan-Philip; Bruckmüller, Henrike; Werk, Anneke N.; Cascorbi, Ingolf; Hoffmann, Wolfgang

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and pr...

  5. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients’ survival. PMID:24380367

  6. Silencing of ABCG2 by MicroRNA-3163 Inhibits Multidrug Resistance in Retinoblastoma Cancer Stem Cells.

    Jia, Ming; Wei, Zhenhua; Liu, Peng; Zhao, Xiaoli

    2016-06-01

    To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma. PMID:27247490

  7. Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study

    Victor Augusto Moraes da Silva

    2015-01-01

    Full Text Available Schizophrenia (SZ is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN, a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at −860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation.

  8. Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study.

    da Silva, Victor Augusto Moraes; Dantas, Marília de Souza; Silva, Leonardo Agostinho de Castro; Carneiro, Juliana Garcia; Schamber-Reis, Bruno Luiz Fonseca

    2015-01-01

    Schizophrenia (SZ) is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN), a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at -860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation. PMID:26526966

  9. Isolation of Side Population Cells and Detection of ABCG2 from SW480

    LIU Hai-guang; PAN Yi-fei; GUO Gui-long; HU Xiao-qu; HUANG Ka-te; ZHANG Xiao-hua

    2007-01-01

    Objective: Side population cells (SP cells) are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods: side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results: SW480 contained 2.29% side population cells. The fraction of side population cells decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction (34.05%) of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. Conclusion: our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.

  10. Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

    Palsamy, Periyasamy [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Ayaki, Masahiko [Shizuoka National Hospital, Saitama (Japan); Elanchezhian, Rajan [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Shinohara, Toshimichi, E-mail: tshinohara@unmc.edu [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer We found significant Keap1 promoter demethylation in diabetic cataractous lenses. Black-Right-Pointing-Pointer Demethylation of Keap1 gene upregulated the expression of Keap1 mRNA and protein. Black-Right-Pointing-Pointer Elevated levels of Keap1 are known to decrease the levels of Nrf2. Black-Right-Pointing-Pointer Thereby, the levels of antioxidant enzymes are suppressed by decreased Nrf2 level. -- Abstract: Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2 Prime deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which

  11. Demethylation of the human eotaxin-3 gene promoter leads to the elevated expression of eotaxin-3

    Lim, Eunjin; Rothenberg, Marc E.

    2013-01-01

    DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. Herein, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue (CTL). The demethy...

  12. Norcantharidin blocks Wnt/β-catenin signaling via promoter demethylation of WIF-1 in glioma.

    Xie, Dajiang; Xie, Jixi; Wan, Yingfeng; Ma, Li; Qi, Xuchen; Wang, Kun; Yang, Shuxu

    2016-04-01

    Glioma is one of the most common primary intracranial tumors, and the prognosis is poor even though much treatment management is employed. Wnt/β-catenin signaling has been reported to be associated with glioma. Norcantharidin (NCTD) is the demethylated analog of cantharidin isolated from blister beetles, and it is reported to possess anticancer activity but less nephrotoxicity than cantharidin. Accordingly, we aimed to investigate NCTD as an anti-neoplastic drug that inhibits the Wnt/β‑catenin pathway via promoter demethylation of Wnt inhibitory factor-1 (WIF-1) in glioma growth in vitro. In the present study, we report that NCTD inhibited cell proliferation, induced apoptosis and cell cycle arrest, and suppressed cell migration and invasion in vitro. Moreover, we observed that the expression levels of WIF-1 mRNA and protein in the NCTD-treated cells were increased significantly compared with these levels in the negative control (NC) cells. Promoter demethylation was observed in the NCTD‑treated cells. In contrast, aberrant methylation was observed in the NC cells. Additionally, more investigation revealed that NCTD suppressed activity of Wnt/β-catenin signaling and transcription of β-catenin/TCF-4. Furthermore, the expression of apoptosis-related proteins Bcl-2 and cleaved caspase-3 indicated significant cell apoptosis. We provide initial evidence that NCTD reactivates WIF-1 from a methylation state, and downregulates canonical Wnt/β-catenin signaling. Our findings revealed that NCTD is effective for glioma in vitro and may be a new therapeutic option in vivo. PMID:26781164

  13. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter. PMID:26907376

  14. Hedgehog Pathway Inhibitor HhAntag691 Is a Potent Inhibitor of ABCG2/BCRP and ABCB1/Pgp

    Yimao Zhang

    2009-01-01

    Full Text Available HhAntag691 (GDC-0449, a low-molecular weight inhibitor of the tumor-promoting hedgehog (Hh signaling pathway, has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors. Here, we show that HhAntag691 inhibits multiple ATP-binding cassette (ABC transporters. ATP-binding cassette transporters are within a family of membrane proteins, the overexpression of which is associated with multidrug resistance, a major impediment to successful cancer treatment. HhAntag691 is a potent inhibitor of two ABC transporters, ABCG2/BCRP and ABCB1/Pgp, and is a mild inhibitor of ABCC1/MRP1. In ABCG2-overexpressing HEK293 cells, HhAntag691 increased retention of the fluorescent ABCG2 substrate BODIPY-prazosin and resensitized these cells to mitoxantrone, an antineoplastic ABCG2 substrate. In Madin-Darby canine kidney II cells engineered to overexpress Pgp or MRP1, HhAntag691 increased the retention of calcein-AM and resensitized them to colchicine. HhAntag691 also resensitized human non-small cell lung carcinoma cells NCI-H460/par and NCI-H460/MX20, which overexpress ABCG2 in response to mitoxantrone, to mitoxantrone, and to topotecan or SN-38. The IC50 values of HhAntag691 for inhibition of ABCG2 and Pgp were ∼1.4 and ∼3.0 µM, respectively. Because ABC transporters are highly expressed at the blood-brain barrier and on many tumor cells, they contribute significantly to treatment failure of many types of cancer, particularly of those within the neuraxis. In addition to its effect on Hh signaling, the ability of HhAntag691 and related compounds to inhibit two key ABC transporters could contribute to their effectiveness in treating malignancies.

  15. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

    Shimada, T.; Inokuchi, K; Nienhuis, A W

    1987-01-01

    The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites ...

  16. BCRP/ ABCG2 in the Placenta: Expression, Function and Regulation

    MAO, QINGCHENG

    2008-01-01

    Knowledge concerning transport of maternally administered drugs across the placental barrier is essential for determining potential toxicity of drugs to the fetus and the value of drug therapy during pregnancy. An important determinant for fetal drug exposure is the expression of efflux transporters in the placenta. Among human tissues, the ATP-binding cassette efflux transporter BCRP (gene symbol ABCG2) is most abundantly expressed in the apical membrane of placental syncytiotrophoblasts. Al...

  17. Mbd2 Promotes Foxp3 Demethylation and T-Regulatory-Cell Function

    Wang, Liqing; Liu, Yujie; Han, Rongxiang; Ulf H Beier; Thomas, Rajan M.; Wells, Andrew D.; Hancock, Wayne W.

    2013-01-01

    Use of Foxp3-positive (Foxp3+) T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmunity, or post-stem cell or -organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3. Conserved CpG dinucleotides in the Treg-specific demethylation region (TSDR) upstream of Foxp3 are demethylated only in stable, thymus-derived Foxp3+ Treg cells. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling com...

  18. Base-oxidant promoted metal-free N-demethylation of arylamines

    VINAYAK BOTLA; CHIRANJEEVI BARREDDI; RAMANA V DAGGUPATI; CHANDRASEKHARAM MALAPAKA

    2016-09-01

    A metal-free oxidative N-demethylation of arylamines with triethylamine as a base and tert-butyl hydroperoxide (TBHP) as oxidant is reported in this paper. The reaction is general, practical, inexpensive, non-toxic, and the method followed is environmentally benign, with moderate to good yields.

  19. Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma

    Sukowati Caecilia HC

    2012-11-01

    Full Text Available Abstract Background The Breast Cancer Resistance Protein (BCRP/ABCG2 is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC in both in vivo and in vitro models with different degree of malignancy. Methods In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot. Results ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 μM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p Conclusions Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors.

  20. Inhibition of ABCG2-mediated transport by protein kinase inhibitors with a bisindolylmaleimide or indolocarbazole structure.

    Robey, Robert W; Shukla, Suneet; Steadman, Kenneth; Obrzut, Tomasz; Finley, Elizabeth M; Ambudkar, Suresh V; Bates, Susan E

    2007-06-01

    ABCG2 is a transporter with potential importance in cancer drug resistance, drug oral absorption, and stem cell biology. In an effort to identify novel inhibitors of ABCG2, we examined the ability of commercially available bisindolylmaleimides (BIM) and indolocarbazole protein kinase inhibitors (PKI) to inhibit ABCG2, given the previous demonstration that the indolocarbazole PKI UCN-01 interacted with the transporter. At a concentration of 10 micromol/L, all of the compounds tested increased intracellular fluorescence of the ABCG2-specific substrate pheophorbide a in ABCG2-transfected HEK-293 cells by 1.3- to 6-fold as measured by flow cytometry; the ABCG2-specific inhibitor fumitremorgin C increased intracellular fluorescence by 6.6-fold. In 4-day cytotoxicity assays, wild-type ABCG2-transfected cells were not more than 2-fold resistant to any of the compounds, suggesting that the PKIs are not significantly transported by ABCG2. BIMs I, II, III, IV, and V, K252c, and arcyriaflavin A were also able to inhibit [(125)I]iodoarylazidoprazosin labeling of ABCG2 by 65% to 80% at 20 micromol/L, compared with a 50% to 70% reduction by 20 micromol/L fumitremorgin C. K252c and arcyriaflavin A were the most potent compounds, with IC(50) values for inhibition of [(125)I]iodoarylazidoprazosin labeling of 0.37 and 0.23 micromol/L, respectively. K252c and arcyriaflavin A did not have any effect on the ATPase activity of ABCG2. Four minimally toxic compounds--BIM IV, BIM V, arcyriaflavin A, and K252c-reduced the relative resistance of ABCG2-transfected cells to SN-38 in cytotoxicity assays. We find that indolocarbazole and BIM PKIs directly interact with the ABCG2 protein and may thus increase oral bioavailability of ABCG2 substrates. PMID:17575116

  1. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression

    Takeo Nakanishi; Douglas D. Ross

    2012-01-01

    Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells.BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract,as well as through the blood-brain,placental,and possibly blood-testis barriers.BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use.Thus,BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs.Because BCRP is also known to be a stem cell marker,its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance,self-renewal (stemness),and invasiveness (aggressiveness),and thereby impart a poor prognosis.Therefore,blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers.Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR.Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α, estrogen receptor, and peroxisome proliferator-activated receptor.Furthermore,alternative promoter usage,demethylation of the BCRP promoter,and histone modificationare likely associated with drug-induced BCRP overexpression in cancer cells.Finally,PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions.These biological events seem involved in a complicated manner.Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with cancer.This review will

  2. Human in vitro induced T regulatory cells and memory T cells share common demethylation of specific FOXP3 promoter region

    Bégin, Philippe; Schulze, Janika; Baron, Udo; Olek, Sven; Rebecca N Bauer; Passerini, Laura; Baccheta, Rosa; Nadeau, Kari C.

    2015-01-01

    Background The FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypo...

  3. ABCG2 Inhibition as a Therapeutic Approach for Overcoming Multidrug Resistance in Cancer

    Maryam Hosseini Hasanabady; Fatemeh Kalalinia

    2016-06-01

    Breast cancer resistance protein (BCRP, ABCP or MXR) / ATP-binding cassette subfamily G member 2 (ABCG2) was characterized as a multidrug resistance efflux transporter in 1998. ABCG2 physiologically acts as a part of a self-defense mechanism for the organism; it enhances eliminating of toxic xenobiotic substances and harmful agents in the intestine, as well as through the blood-brain barrier and placental. ABCG2 recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and new targeted small therapeutic molecules in clinical usage. Development of ABCG2 inhibitors for clinical usage may allow increased penetration of therapeutic agents into sanctuary sites and increased their intestinal absorption. In this report, we review the mechanisms that modulate MDR mediated by the ABC transporter ABCG2 in normal and cancer cells by different levels including, epigenetic modifications, transcriptional, posttranscriptional, translation and posttranslational regulation. Some clinical applications of ABCG2 inhibitors, also is explained.

  4. PSEN1 promoter demethylation in hyperhomocysteinemic TgCRND8 mice is the culprit, not the consequence.

    Fuso, Andrea; Cavallaro, Rosaria A; Cavallaroa, Rosaria A; Nicolia, Vincenzina; Scarpa, Sigfrido

    2012-06-01

    In recent years, in parallel with the growing awareness of the multifactorial nature of Late Onset Alzheimer's Disease, the possibility that epigenetic mechanisms could be involved in the onset and/or progression of the pathology assumed an increasingly intriguing and leading role in Alzheimer's research. Today, many scientific reports indicate the existence of an epigenetic drift during ageing, in particular in Alzheimer's subjects. At the same time, experimental evidences are provided with the aim to demonstrate the causative or consequential role of epigenetic mechanisms. Our research group was involved in the last ten years in studying DNA methylation, the main epigenetic modification, in relationship to altered one-carbon metabolism (namely high homocysteine and low B vitamins levels), in Alzheimer's experimental models. Our previous findings about the demethylation of Presenilin1 gene promoter in nutritionally-induced hyperhomocysteinemia in a transgenic mouse model clearly demonstrated that Presenilin1 is regulated by DNA methylation. One of the open questions raised by our studies was if the observed demethylation was solely due to the induced imbalance of one-carbon metabolism or could be a response to the massive deposition of amyloid plaques in transgenic mice. Here we analyzed old (10 months) mice under standard diet in order to evidence possible changes in Presenilin1 promoter methylation in transgenic (TgCRND8 mice, carrying a double-mutated human APP transgene) vs. wt mice (129Sv) after prolonged exposure to amyloid. We found no differences in Presenilin1 methylation despite a slight increase in gene expression; these results suggest that amyloid production is not responsible for Presenilin1 demethylation in TgCRND8 mice brain. PMID:22272624

  5. Norcantharidin inhibits Wnt signal pathway via promoter demethylation of WIF-1 in human non-small cell lung cancer.

    Xie, Junran; Zhang, Yaping; Hu, Xuming; Lv, Ran; Xiao, Dongju; Jiang, Li; Bao, Qi

    2015-05-01

    Wingless-type (Wnt) family of secreted glycoproteins is a group of signal molecules implicated in oncogenesis. Abnormal activation of Wnt signal pathway is associated with a variety of human cancers, including non-small cell lung cancer (NSCLC). Wnt antagonists, such as the secreted frizzled-related protein (SFRP) family, Wnt inhibitory factor-1 (WIF-1) and cerberus, inhibit Wnt signal pathway by directly binding to Wnt molecules. Norcantharidin (NCTD) is known to possess anticancer activity but less nephrotoxicity than cantharidin. In this study, we found that NCTD inhibited cell proliferation, induced apoptosis, arrested cell cycle and suppressed cell invasion/migration in vitro. Additionally, Wnt signal pathway transcription was also suppressed. NCTD treatment blocked cytoplasmic translocation of beta-catenin into the nucleus. Alterations of apoptosis-related proteins, such as Bax, cleaved caspase-3 (pro-apoptotic) and Bcl-2 (anti-apoptotic), had been detected. Furthermore, the expression levels of WIF-1 and SFRP1 were significantly increased in NCTD-treated groups compared with negative control (NC) groups. Abnormal methylation was observed in NC groups, while NCTD treatment promoted WIF-1 demethylation. The present study revealed that NCTD activated WIF-1 via promoter demethylation, inhibiting the canonical Wnt signal pathway in NSCLC, which may present a new therapeutic target in vivo. PMID:25814287

  6. Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

    Yuan Li; Qiong Xia; Hongping Kou; Dan Wang; Xiuyun Lin; Ying Wu; Chunming Xu; Shaochen Xing

    2011-01-01

    Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily.Expression of Pib was low under non-challenged conditions,but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea,thereby conferring resistance to the pathogen.It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question.We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied.Surprisingly,induced expression of Pib by M.grisea infection did not entail its promoter demethylation,and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wildtype plants.Accordingly,the blast disease-resistance was compromised in the 5’-azaC-treated plants relative to wild-type.In contrast,the disease susceptibility was not affected by the 5’-azaC treatment in another two rice cultivars that did not contain the Pib gene,ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance.Taken together,our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M.grisea infection,and its conferred resistance to the pathogen.

  7. Hyperuricemia in acute gastroenteritis is caused by decreased urate excretion via ABCG2.

    Matsuo, Hirotaka; Tsunoda, Tomoyuki; Ooyama, Keiko; Sakiyama, Masayuki; Sogo, Tsuyoshi; Takada, Tappei; Nakashima, Akio; Nakayama, Akiyoshi; Kawaguchi, Makoto; Higashino, Toshihide; Wakai, Kenji; Ooyama, Hiroshi; Hokari, Ryota; Suzuki, Hiroshi; Ichida, Kimiyoshi; Inui, Ayano; Fujimori, Shin; Shinomiya, Nariyoshi

    2016-01-01

    To clarify the physiological and pathophysiological roles of intestinal urate excretion via ABCG2 in humans, we genotyped ABCG2 dysfunctional common variants, Q126X (rs72552713) and Q141K (rs2231142), in end-stage renal disease (hemodialysis) and acute gastroenteritis patients, respectively. ABCG2 dysfunction markedly increased serum uric acid (SUA) levels in 106 hemodialysis patients (P = 1.1 × 10(-4)), which demonstrated the physiological role of ABCG2 for intestinal urate excretion because their urate excretion almost depends on intestinal excretion via ABCG2. Also, ABCG2 dysfunction significantly elevated SUA in 67 acute gastroenteritis patients (P = 6.3 × 10(-3)) regardless of the degree of dehydration, which demonstrated the pathophysiological role of ABCG2 in acute gastroenteritis. These findings for the first time show ABCG2-mediated intestinal urate excretion in humans, and indicates the physiological and pathophysiological importance of intestinal epithelium as an excretion pathway besides an absorption pathway. Furthermore, increased SUA could be a useful marker not only for dehydration but also epithelial impairment of intestine. PMID:27571712

  8. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells

    Ozvegy, C; Litman, Thomas; Szakács, G; Nagy, Z; Bates, S; Váradi, A; Sarkadi, B

    2001-01-01

    membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N...

  9. Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues

    Fetsch, Patricia A; Abati, Andrea; Litman, Thomas;

    2006-01-01

    determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a...... modified avidin-biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was...... ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates....

  10. Porphyrin homeostasis maintained by ABCG2 regulates self-renewal of embryonic stem cells.

    Jimmy Susanto

    Full Text Available BACKGROUND: Under appropriate culture conditions, undifferentiated embryonic stem (ES cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined. METHODOLOGY/PRINCIPAL FINDINGS: Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC, a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of gammaH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment. CONCLUSIONS/SIGNIFICANCE: The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and gammaH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.

  11. Characterization of the role of ABCG2 as a bile acid transporter in liver and placenta.

    Blazquez, Alba G; Briz, Oscar; Romero, Marta R; Rosales, Ruben; Monte, Maria J; Vaquero, Javier; Macias, Rocio I R; Cassio, Doris; Marin, Jose J G

    2012-02-01

    ABCG2 is involved in epithelial transport/barrier functions. Here, we have investigated its ability to transport bile acids in liver and placenta. Cholylglycylamido fluorescein (CGamF) was exported by WIF-B9/R cells, which do not express the bile salt export pump (BSEP). Sensitivity to typical inhibitors suggested that CGamF export was mainly mediated by ABCG2. In Chinese hamster ovary (CHO cells), coexpression of rat Oatp1a1 and human ABCG2 enhanced the uptake and efflux, respectively, of CGamF, cholic acid (CA), glycoCA (GCA), tauroCA, and taurolithocholic acid-3-sulfate. The ability of ABCG2 to export these bile acids was confirmed by microinjecting them together with inulin in Xenopus laevis oocytes expressing this pump. ABCG2-mediated bile acid transport was inhibited by estradiol 17β-d-glucuronide and fumitremorgin C. Placental barrier for bile acids accounted for 14-fold increased maternal cholanemia induced by obstructive cholestasis in pregnant rats. In rat placenta, the expression of Abcg2, which was much higher than that of Bsep, was not affected by short-term cholestasis. In pregnant rats, fumitremorgin C did not affect uptake/secretion of GCA by the liver but inhibited its fetal-maternal transfer. Compared with wild-type mice, obstructive cholestasis in pregnant Abcg2(-/-) knockout mice induced similar bile acid accumulation in maternal serum but higher accumulation in placenta, fetal serum, and liver. In conclusion, ABCG2 is able to transport bile acids. The importance of this function depends on the relative expression in the same epithelium of other bile acid exporters. Thus, ABCG2 may play a key role in bile acid transport in placenta, as BSEP does in liver. PMID:22096226

  12. ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity

    AttilioDi Pietro

    2013-11-01

    Full Text Available ABCG2 is a key human ATP-Binding Cassette (ABC transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated GSH efflux and, upon depletion of intracellular glutathione (GSH, induce “collateral sensitivity” leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and 3H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.

  13. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    La, Honggui

    2011-09-06

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

  14. Effect of Walker A mutation (K86M) on oligomerization and surface targeting of the multidrug resistance transporter ABCG2

    Henriksen, Ulla Birk; Gether, Ulrik; Litman, Thomas

    The ATP binding cassette (ABC) half-transporter ABCG2 (MXR/BCRP/ABCP) is associated with mitoxantrone resistance accompanied by cross-resistance to a broad spectrum of cytotoxic drugs. Here we investigate the functional consequences of mutating a highly conserved lysine in the Walker A motif of the...... nucleotide binding domain (NBD) known to be critical for ATP binding and/or hydrolysis in ABC transporters. The mutant (ABCG2-K86M) was inactive as expected but was expressed at similar levels as the wild-type (wt) protein. The mutation did not affect the predicted oligomerization properties of the...... transporter; hence, co-immunoprecipitation experiments using differentially tagged transporters showed evidence for oligomerization of both ABCG2-wt and of ABCG2-wt with ABCG2-K86M. We also obtained evidence that both ABCG2-wt and ABCG2-K86M exist in the cells as disulfide-linked dimers. Moreover, measurement...

  15. Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity

    Nagai, Shinjiro; Takenaka, Kazumasa; Nachagari, Deepa; Rose, Charles; Domoney, Kali; Sun, Daxi; Sparreboom, Alex; Schuetz, John D.

    2011-01-01

    Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK ...

  16. Expression of ABCG2 (BCRP in mouse models with enhanced erythropoiesis

    Gladys Oluyemisi Latunde-Dada

    2014-06-01

    Full Text Available Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2, an ATP Binding Cassette G-member protein has been shown to possess porhyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ. Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1 levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification.

  17. Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

    Hsu Anna

    2011-10-01

    Full Text Available Abstract Sulforaphane (SFN, an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs, especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

  18. Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model.

    Halwachs, Sandra; Schäfer, Ingo; Kneuer, Carsten; Seibel, Peter; Honscha, Walther

    2016-08-15

    In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim, chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B>A) directed transport of [(14)C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates

  19. Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

    Henriksen, Ulla Birk; Fog, Jacob U; Litman, Thomas; Gether, Ulrik

    2005-01-01

    cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band....... Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer...

  20. Associations Between ABCG2 Gene Polymorphisms and Isolated Septal Defects in a Han Chinese Population

    Wang, Chuan; Xie, Liang; Li, Huaying; Li, Yifei; Mu, Dezhi; Zhou, Rong; Liu, Ruiqi; Zhou, Kaiyu; Hua, Yimin

    2014-01-01

    Breast cancer resistance protein (BCRP) in the placenta, encoded by the ABCG2 gene in humans, plays an essential role in regulating fetal exposure to toxicants and the maintenance of cellular folic acid homeostasis. This study aimed at exploring the associations between 421C>A and 34G>A polymorphisms within the ABCG2 gene of the children and isolated septal defects in a Han Chinese population. An age- and gender-matched case-control study involving 210 pairs was conducted. Genotyping of the A...

  1. ABCG2 variant has opposing effects on onset ages of Parkinson's disease and gout

    Matsuo, Hirotaka; TOMIYAMA, Hiroyuki; Satake, Wataru; Chiba, Toshinori; Onoue, Hiroyuki; Kawamura, Yusuke; Nakayama, Akiyoshi; Shimizu, Seiko; Sakiyama, Masayuki; Funayama, Manabu; Nishioka, Kenya; SHIMIZU, TORU; Kaida, Kenichi; Kamakura, Keiko; Toda, Tatsushi

    2015-01-01

    Uric acid (urate) has been suggested to play a protective role in Parkinson's disease onset through its antioxidant activity. Dysfunction of ABCG2, a high-capacity urate exporter, is a major cause for early-onset gout based on hyperuricemia. In this study, the effects of a dysfunctional ABCG2 variant (Q141K, rs2231142) were analyzed on the ages at onset of gout patients (N = 507) and Parkinson's disease patients (N = 1015). The Q141K variant hastened the gout onset (P = 0.0027), but significa...

  2. Demethylation profile of the TNF-α promoter gene is associated with high expression of this cytokine in Dengue virus patients.

    Gomes, Alessandra Vilas Boas Terra; de Souza Morais, Stella Maria; Menezes-Filho, Sergio Luiz; de Almeida, Luiz Gustavo Nogueira; Rocha, Raissa Prado; Ferreira, Jaqueline Maria Siqueira; Dos Santos, Luciana Lara; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-08-01

    Dengue is the most prevalent arthropod-borne viral illness in humans. The overexpression of cytokines by Dengue virus (DENV) infected cells is associated with the most severe forms of the disease. Unmethylated CpG islands are related to a transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that inhibit gene expression. Several studies have described the importance of epigenetic events in the regulation and expression of many cytokines. The purpose of the present study was to evaluate the methylation status of the IFN-γ and TNF-α promoters in DNA extracted from dengue infected patients using methylation-specific polymerase chain reaction. A high frequency of demethylation was observed in the TNF-α promoter of DENV infected patients when compared to non-infected controls. The patients with an unmethylated profile showed higher expression of TNF-α mRNA than patients with the methylated status. No difference was found in the methylation frequency between the two analyzed groups regarding the IFN-γ promoter or in the expression of IFN-γ transcripts. The present study provides the first association of TNF-α promoter demethylation in DENV infected individuals and demonstrates a correlation between the methylation status of the region analyzed and the expression of TNF-α transcripts in DENV infected patients. J. Med. Virol. 88:1297-1302, 2016. © 2016 Wiley Periodicals, Inc. PMID:26792115

  3. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  4. ABCG2 regulates self-renewal and stem cell marker expression but not tumorigenicity or radiation resistance of glioma cells

    Wee, Boyoung; Pietras, Alexander; Ozawa, Tatsuya; Bazzoli, Elena; Podlaha, Ondrej; Antczak, Christophe; Westermark, Bengt; Nelander, Sven; Uhrbom, Lene; Forsberg-Nilsson, Karin; Djaballah, Hakim; Michor, Franziska; Holland, Eric C.

    2016-01-01

    Glioma cells with stem cell traits are thought to be responsible for tumor maintenance and therapeutic failure. Such cells can be enriched based on their inherent drug efflux capability mediated by the ABC transporter ABCG2 using the side population assay, and their characteristics include increased self-renewal, high stem cell marker expression and high tumorigenic capacity in vivo. Here, we show that ABCG2 can actively drive expression of stem cell markers and self-renewal in glioma cells. Stem cell markers and self-renewal was enriched in cells with high ABCG2 activity, and could be specifically inhibited by pharmacological and genetic ABCG2 inhibition. Importantly, despite regulating these key characteristics of stem-like tumor cells, ABCG2 activity did not affect radiation resistance or tumorigenicity in vivo. ABCG2 effects were Notch-independent and mediated by diverse mechanisms including the transcription factor Mef. Our data demonstrate that characteristics of tumor stem cells are separable, and highlight ABCG2 as a potential driver of glioma stemness. PMID:27456282

  5. Human ABC transporter ABCG2/BCRP expression in chemoresistance: basic and clinical perspectives for molecular cancer therapeutics

    Noguchi K

    2014-02-01

    Full Text Available Kohji Noguchi, Kazuhiro Katayama, Yoshikazu Sugimoto Division of Chemotherapy, Faculty of Pharmacy, Keio University, Tokyo, Japan Abstract: Adenine triphosphate (ATP-binding cassette (ABC transporter proteins, such as ABCB1/P-glycoprotein (P-gp and ABCG2/breast cancer resistance protein (BCRP, transport various structurally unrelated compounds out of cells. ABCG2/BCRP is referred to as a “half-type” ABC transporter, functioning as a homodimer, and transports anticancer agents such as irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38, gefitinib, imatinib, methotrexate, and mitoxantrone from cells. The expression of ABCG2/BCRP can confer a multidrug-resistant phenotype on cancer cells and affect drug absorption, distribution, metabolism, and excretion in normal tissues, thus modulating the in vivo efficacy of chemotherapeutic agents. Clarification of the substrate preferences and structural relationships of ABCG2/BCRP is essential for our understanding of the molecular mechanisms underlying its effects in vivo during chemotherapy. Its single-nucleotide polymorphisms are also involved in determining the efficacy of chemotherapeutics, and those that reduce the functional activity of ABCG2/BCRP might be associated with unexpected adverse effects from normal doses of anticancer drugs that are ABCG2/BCRP substrates. Importantly, many recently developed molecular-targeted cancer drugs, such as the tyrosine kinase inhisbitors, imatinib mesylate, gefitinib, and others, can also interact with ABCG2/BCRP. Both functional single-nucleotide polymorphisms and inhibitory agents of ABCG2/BCRP modulate the in vivo pharmacokinetics and pharmacodynamics of these molecular cancer treatments, so the pharmacogenetics of ABCG2/BCRP is an important consideration in the application of molecular-targeted chemotherapies. Keywords: kinase inhibitor, SNP, single-nucleotide polymorphisms, molecular target

  6. Antibody validation and scoring guidelines for ABCG2 immunohistochemical staining in formalin-fixed paraffin-embedded colon cancer tissue

    Cederbye, Camilla Natasha; Palshof, Jesper Andreas; Hansen, Tine Plato;

    2016-01-01

    cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we...... cytoplasmic signal. Intra-tumor heterogeneity in ABCG2 immunoreactivity was observed; however, statistical analyses of tissue microarrays (TMAs) and the corresponding whole sections from primary tumors of 57 metastatic CRC patients revealed a strong positive correlation between maximum TMA scores and whole...

  7. Brief Report: High Peak Level of Plasma Raltegravir Concentration in Patients With ABCB1 and ABCG2 Genetic Variants.

    Tsuchiya, Kiyoto; Hayashida, Tsunefusa; Hamada, Akinobu; Oka, Shinichi; Gatanaga, Hiroyuki

    2016-05-01

    Raltegravir was recently identified to be a substrate of ATP-binding cassette transporter B1 (ABCB1) and G2 (ABCG2), which are efflux transporters and expressed in the intestines. We analyzed the relations between plasma raltegravir concentrations and single nucleotide polymorphism of ABCB1 and ABCG2 genes. The peak plasma concentration of raltegravir was significantly higher in the patients with ABCB1 4036 AG/GG and ABCG2 421 CA/AA than in other genotype holders (P = 0.0052), though no difference was identified in trough raltegravir concentrations, which may be explained by reduced expression of efflux transporters in intestine by these genetic variants. PMID:27097364

  8. BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma Cells to Sorafenib

    Huang, Wei-Chien; Hsieh, Yi-Ling; Hung, Chao-Ming; Chien, Pei-Hsuan; Chien, Yu-Fong; Chen, Lei-Chin; Tu, Chih-Yen; Chen, Chia-Hung; Hsu, Sheng-Chieh; Lin, Yueh-Ming; Chen, Yun-Ju

    2013-01-01

    The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine ...

  9. DGAT1 and ABCG2 polymorphism in Indian cattle (Bos indicus and buffalo (Bubalus bubalis breeds

    Mishra Bina

    2006-11-01

    Full Text Available Abstract Background Indian cattle (Bos indicus and riverine buffalo (Bubalus bubalis give a poor yield of milk but it has a high fat and protein percentage compared to taurine cattle. The identification of QTLs (Quantitative Trait Loci on BTA14 and BTA6 and its subsequent fine mapping has led to identification of two non conservative mutations affecting milk production and composition. Our objective was to estimate the frequency of K232A (DGAT1 – diacylglycerol – acyltransferase 1 and Y581S (ABCG2 – ATP binding cassette sub family G member 2 polymorphisms in diverse cattle and buffalo breeds of India having large variation in terms of milk production. Results We screened the reported missense mutations in six cattle and five buffalo breeds. The DGAT1K and ABCG2Y alleles were found to be fixed in Indian cattle and buffalo breeds studied. Conclusion This study provides an indirect evidence that all the Indian cattle and buffalo breeds have fixed alleles with respect to DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent.

  10. Overcoming multidrug resistance via photodestruction of ABCG2-rich extracellular vesicles sequestering photosensitive chemotherapeutics.

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs and topotecan resulted in intravesicular formation of reactive oxygen species (ROS and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently

  11. Transcriptional activation of the Epstein-Barr virus latency C promoter after 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.

    Robertson, K D; Hayward, S D; Ling, P D; Samid, D; Ambinder, R F

    1995-01-01

    The Epstein-Barr Virus (EBV) latency C promoter (Cp) is the origin of transcripts for six viral proteins. The promoter is active in lymphoblastoid B-cell lines but silent in many EBV-associated tumors and tumor cell lines. In these latter cell lines, the viral episome is hypermethylated in the vicinity of this promoter. We show that in such a cell line (Rael, a Burkitt's lymphoma line), 5-azacytidine inhibits DNA methyltransferase, brings about demethylation of EBV genomes, activates Cp transcription, and induces the expression of EBNA-2. Investigation of the phenomenon demonstrates the importance of the methylation status of a particular CpG site for the regulation of the Cp: (i) genomic sequencing shows that this site is methylated when the Cp is inactive and is not methylated when the promoter is active; (ii) methylation or transition mutation at this site abolishes complex formation with a cellular binding activity (CBF2) as determined by electrophoretic mobility shift analyses, competition binding analyses, and DNase I footprinting; and (iii) a single C --> T transition mutation at this site is associated with a marked reduction (> 50-fold) of transcriptional activity in a reporter plasmid. Thus, the CBF2 binding activity is shown to be methylation sensitive and crucial to EBNA-2-mediated activation of the Cp. PMID:7565767

  12. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line.

    Song, Deye; Ni, Jiangdong; Xie, Hongming; Ding, Muliang; Wang, Jun

    2014-05-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  13. DNA demethylation in the PTEN gene promoter induced by 5-azacytidine activates PTEN expression in the MG-63 human osteosarcoma cell line

    SONG, DEYE; NI, JIANGDONG; XIE, HONGMING; DING, MULIANG; WANG, JUN

    2014-01-01

    This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the

  14. Re-expression of RASSF1A by 5-Aza-CdR Induced Demethylation of the Promoter Region in Human Biliary Tract Carcinoma Cells

    ZUO Shi; CHEN Yongjun; XU Lining; TANG Qibin; ZOU Shengquan

    2007-01-01

    Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation,5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

  15. Active DNA Demethylation Mediated by DNA Glycosylases

    Zhu, Jian-Kang

    2009-01-01

    Active DNA demethylation is involved in many vital developmental and physiological processes of plants and animals. Recent genetic and biochemical studies in Arabidopsis have demonstrated that a subfamily of DNA glycosylases function to promote DNA demethylation through a base excision-repair pathway. These specialized bifunctional DNA glycosylases remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, resulting in a gap that is then filled with an unmethylated ...

  16. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  17. Multidrug Transporter ABCG2/Breast Cancer Resistance Protein Secretes Riboflavin (Vitamin B2) into Milk▿

    van Herwaarden, Antonius E.; Wagenaar, Els; Merino, Gracia; Jonker, Johan W.; Rosing, Hilde; Beijnen, Jos H.; Schinkel, Alfred H.

    2006-01-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B2) into milk, thus supplying the young with this important nutrient. In Bcrp1−/− mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1−/− pups showed no riboflavin deficiency due to concomitant mi...

  18. Effect of bovine ABCG2 polymorphism Y581S SNP on secretion into milk of enterolactone, riboflavin and uric acid.

    Otero, J A; Miguel, V; González-Lobato, L; García-Villalba, R; Espín, J C; Prieto, J G; Merino, G; Álvarez, A I

    2016-02-01

    The ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) is an efflux protein involved in the bioavailability and milk secretion of endogenous and exogenous compounds, actively affecting milk composition. A limited number of physiological substrates have been identified. However, no studies have reported the specific effect of this polymorphism on the secretion into milk of compounds implicated in milk quality such as vitamins or endogenous compounds. The bovine ABCG2 Y581S polymorphism is described as a gain-of-function polymorphism that increases milk secretion and decreases plasma levels of its substrates. This work aims to study the impact of Y581S polymorphism on plasma disposition and milk secretion of compounds such as riboflavin (vitamin B2), enterolactone, a microbiota-derived metabolite from the dietary lignan secoisolariciresinol and uric acid. In vitro transport of these compounds was assessed in MDCK-II cells overexpressing the bovine ABCG2 (WT-bABCG2) and its Y581S variant (Y581S-bABCG2). Plasma and milk levels were obtained from Y/Y homozygous and Y/S heterozygous cows. The results show that riboflavin was more efficiently transported in vitro by the Y581S variant, although no differences were noted in vivo. Both uric acid and enterolactone were substrates in vitro of the bovine ABCG2 variants and were actively secreted into milk with a two-fold increase in the milk/plasma ratio for Y/S with respect to Y/Y cows. The in vitro ABCG2-mediated transport of the drug mitoxantrone, as a model substrate, was inhibited by enterolactone in both variants, suggesting the possible in vivo use of this enterolignan to reduce ABCG2-mediated milk drug transfer in cows. The Y581S variant was inhibited to a lesser extent probably due to its higher transport capacity. All these findings point to a significant role of the ABCG2 Y581S polymorphism in the milk disposition of enterolactone and the endogenous molecules riboflavin and uric acid

  19. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood-Brain Barrier.

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier-Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M; Langer, O

    2016-08-01

    ABCB1 and ABCG2 work together at the blood-brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([(11) C]elacridar and [(11) C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single-nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high-dose tariquidar. In contrast to the ABCB1-selective substrate (R)-[(11) C]verapamil, [(11) C]elacridar and [(11) C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [(11) C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. PMID:26940368

  20. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood–Brain Barrier

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier‐Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M

    2016-01-01

    ABCB1 and ABCG2 work together at the blood–brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([11C]elacridar and [11C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single‐nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high‐dose tariquidar. In contrast to the ABCB1‐selective substrate (R)‐[11C]verapamil, [11C]elacridar and [11C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [11C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. PMID:26940368

  1. Expression of ABCG2 and Bmi-1 in oral potentially malignant lesions and oral squamous cell carcinoma

    Early diagnosis is vital for effective treatment of oral squamous cell carcinoma (OSCC). The optimal time for clinical intervention is prior to malignancy when patients present with oral potentially malignant lesions such as leukoplakia or erythroplakia. Transformation rates for oral dysplasia vary greatly and more rigorous methods are needed to predict the malignant potential of oral lesions. We hypothesized that the expression of two putative stem cell markers, ABCG2 and Bmi-1, would correlate with disease severity for non diseased, potentially malignant and OSCC specimens and cell lines derived from an equivalent range of tissues. We compared immunoreactive protein and relative gene expression of ABCG2 and Bmi-1 in eight cell lines derived from source tissues ranging in disease severity from normal (OKF6-TERT2) through mild and moderate/severe dysplasia (DOK, POE-9n) to OSCC (PE/CA-PJ15, SCC04, SCC25, SCC09, SCC15). We also analyzed immunoreactive protein expression of ABCG2 and Bmi-1 in 189 tissue samples with the same range of disease severity. A trend between oral lesion severity to ABCG2 and Bmi-1 immunostain intensity was observed. Flow cytometry of oral cell lines confirmed this trend and gave good correlation with RT-PCR results for ABCG2 (r = 0.919, P = 0.001; Pearson) but not Bmi-1 (r = −0.311). The results provide evidence of increased density of ABCG2 and Bmi-1-positive populations in malignant and oral potentially malignant lesions and derived cell lines, but that intragroup variability within IHC, flow cytometry, and RT-PCR results compromise the diagnostic potential of these techniques for discriminating oral dysplasia from normal tissue or OSCC

  2. Isolation of side population cells from gallbladder carcinoma of human being and the expression of ABCG2 gene%人胆囊癌SP细胞的分离及ABCG2的表达

    2007-01-01

    Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Methods:Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence chemistry, western blot and flow cytometry techniques. Results:A very small fraction cells (0.64 ± 0.08%) were isolated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ±1.61%). Conclusion: The side population cells that had the potency of stem ceils existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.

  3. OGG1 is essential in oxidative stress induced DNA demethylation.

    Zhou, Xiaolong; Zhuang, Ziheng; Wang, Wentao; He, Lingfeng; Wu, Huan; Cao, Yan; Pan, Feiyan; Zhao, Jing; Hu, Zhigang; Sekhar, Chandra; Guo, Zhigang

    2016-09-01

    DNA demethylation is an essential cellular activity to regulate gene expression; however, the mechanism that triggers DNA demethylation remains unknown. Furthermore, DNA demethylation was recently demonstrated to be induced by oxidative stress without a clear molecular mechanism. In this manuscript, we demonstrated that 8-oxoguanine DNA glycosylase-1 (OGG1) is the essential protein involved in oxidative stress-induced DNA demethylation. Oxidative stress induced the formation of 8-oxoguanine (8-oxoG). We found that OGG1, the 8-oxoG binding protein, promotes DNA demethylation by interacting and recruiting TET1 to the 8-oxoG lesion. Downregulation of OGG1 makes cells resistant to oxidative stress-induced DNA demethylation, while over-expression of OGG1 renders cells susceptible to DNA demethylation by oxidative stress. These data not only illustrate the importance of base excision repair (BER) in DNA demethylation but also reveal how the DNA demethylation signal is transferred to downstream DNA demethylation enzymes. PMID:27251462

  4. Toxicokinetics of the food-toxin IQ in human placental perfusion is not affected by ABCG2 or xenobiotic metabolism

    Immonen, E; Kummu, M; Petsalo, A;

    2010-01-01

    M, n = 6) or Ko143 (specific inhibitor of ABCG2, 2 muM, n = 4) to study the role of ABCG2 inhibition in transfer while in Denmark perfusions were performed with (14)C-IQ alone. Critical parameters (leak from fetal to maternal circulation, pH values, blood gases, glucose consumption, the production of h......CG hormone and transport of antipyrine) were analyzed during the perfusions. (14)C-IQ on maternal and fetal sides was determined by liquid scintillation counting. In Finland IQ and its metabolites in final perfusates were determined also by LC/TOF-MS. ABCG2 expression and EROD activity (CYP1A1/2) were...... analyzed from perfused tissues. (14)C-IQ was easily transferred through the placenta from maternal to fetal side in both laboratories. Neither significant EROD activity nor IQ metabolites were found in placentas from non-smoking mothers. Inhibition of ABCG2 by GF120918 (FM-ratio of IQ 0.95) or Ko143 (FM...

  5. A gene-wide investigation on polymorphisms in the ABCG2/BRCP transporter and susceptibility to colorectal cancer

    Campa, D.; Pardini, Barbara; Naccarati, Alessio; Vodičková, Ludmila; Novotný, J.; Försti, A.; Hemminki, K.; Barale, R.; Vodička, Pavel; Canzian, F.

    2008-01-01

    Roč. 645, 1-2 (2008), s. 56-60. ISSN 0027-5107 R&D Projects: GA ČR GA310/07/1430 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : ABCG2 * Transporter * Colorectal cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.198, year: 2008

  6. High ABCC2 and Low ABCG2 Gene Expression Are Early Events in the Colorectal Adenoma-Carcinoma Sequence

    Andersen, Vibeke; Vogel, Lotte K.; Kopp, Tine Iskov;

    2015-01-01

    Development of colorectal cancer (CRC) may result from a dysfunctional interplay between diet, gut microbes and the immune system. The ABC transport proteins ABCB1 (P-glycoprotein, Multidrug resistance protein 1, MDR1), ABCC2 (MRP2) and ABCG2 (BCRP) are involved in transport of various compounds...

  7. Correlation of HIF-2α, ABCG2 and OCT-4 with chemotherapy resistance in human gastric cancer

    Hong-mei ZHANG

    2015-11-01

    Full Text Available Objective To investigate the correlation of HIF-2α, ABCG2 and OCT-4 with chemotherapy resistant gastric cancer in humans. Methods Fifty-two patients who were confirmed to have advanced gastric cancer with the aid of electronic endoscopy and pathology in the Department of Gastroenterology, Affiliated Hospital of Weifang Medical College, were enrolled in the study. According to the effect of FOL-FOX4 chemotherapy that these patients had experienced, they were divided into three groups: CR+PR (complete remission+partial remission group, SD (stable disease group and PD (progressive disease group. The expression levels of HIF-2α, ABCG2, and OCT-4 mRNA and protein were assessed in different groups by using RT-PCR and immunocytochemistry. Results Two patients achieved CR , 19 achieved PR , 25 showed SD, and 6 showed PD. In other words, CR+PR were seen in 21 patients (40.4%, SD in 25(48.1%, PD in 6(11.5%. In CR+PR group, the expression levels of HIF-2α, ABCG2 and OCT4 mRNA and protein were low, but the above mentioned expressions were significantly increased in SD group and PD group. The expression levels of HIF-2α, ABCG2 and Oct-4 mRNA and protein were highest in the PD group, lower in the SD group, and lowest in the CR + PR groups (all P<0.05. Conclusions The expression of the markers HIF-2α, ABCG2 and OCT4 in human tumor tissues is related to the effect of chemotherapy for gastric cancer. A high expression of tumor markers is perhaps the main reason for low efficacy of chemotherapy due to drug resistance. DOI: 10.11855/j.issn.0577-7402.2015.10.09

  8. Effect of bovine ABCG2 Y581S polymorphism on concentrations in milk of enrofloxacin and its active metabolite ciprofloxacin.

    Otero, J A; García-Mateos, D; de la Fuente, A; Prieto, J G; Álvarez, A I; Merino, G

    2016-07-01

    The ATP-binding cassette transporter G2 (ABCG2) is involved in the secretion of several drugs into milk. The bovine Y581S ABCG2 polymorphism increases the secretion into milk of the fluoroquinolone danofloxacin in Holstein cows. Danofloxacin and enrofloxacin are the fluoroquinolones most widely used in veterinary medicine. Both enrofloxacin (ENRO) and its active metabolite ciprofloxacin (CIPRO) reach milk at relatively high concentrations. The aim of this work was to study the effect of the bovine Y581S ABCG2 polymorphism on in vitro transport as well as on concentrations in plasma and in milk of ENRO and CIPRO. Experiments using cells overexpressing bovine ABCG2 showed the effects of ABCG2 on the transport of CIPRO, demonstrating more efficient in vitro transport of this antimicrobial by the S581 variant as compared with the Y581 variant. Animal studies administering 2.5mg/kg of ENRO subcutaneously to Y/Y 581 and Y/S 581 cows revealed that concentrations in plasma of ENRO and CIPRO were significantly lower in Y/S animals. Regardless of the genotype, the antimicrobial profile in milk after the administration of ENRO was predominantly of CIPRO. With respect to the genotype effects on the amounts of drugs present in milk, AUC0-24 values were more than 1.2 times higher in Y/S cows for ENRO and 2.2 times for CIPRO, indicating a greater capacity of Y581S to transfer these drugs into milk. These results emphasize the clinical relevance of this polymorphism as a factor affecting the concentrations in plasma and in milk of drugs of importance in veterinary medicine. PMID:27157572

  9. Tyrosine kinase inhibitors influence ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway.

    Pick, Anne; Wiese, Michael

    2012-04-01

    Multidrug resistance observed in cancer chemotherapy is commonly attributed to overexpression of efflux transporter proteins. These proteins act as ATP-dependent drug efflux pumps, actively extruding chemotherapeutic agents from cells and causing a decrease in intracellular drug accumulation. Besides the well-recognized role of P-glycoprotein (P-gp, ABCB1), the breast cancer resistance protein (BCRP, ABCG2) is becoming increasingly accepted as playing an important role in multidrug resistance. In contrast to P-glycoprotein, only a few inhibitors of ABCG2 are known. According to the literature, tyrosine kinase inhibitors (TKIs) can be considered to be broad-spectrum inhibitors, interacting with ABCB1, ABCC1 and ABCG2. Here, we investigated seven different TKIs, gefitinib, erlotinib, AG1478, PD158780, PD153035, nilotinib and imatinib, for their potential to restore ABCG2 sensitivity to cells. Furthermore, we analyzed the alteration of ABCG2 expression caused by TKIs and demonstrated that EGFR inhibitors such as gefitinib and PD158780 reduced both total and surface expression of ABCG2 in EGRF-positive MDCK BCRP cells by interaction with the PI3K/Akt signaling pathway. The reduced ABCG2 content led to an increased effect of XR9577, a well-known ABCG2 modulator, lowering the concentration required for half maximal inhibition. On the other hand, BCR-ABL inhibitors had no influence on ABCG2 expression and modulator activity. Interestingly, a combination of an EGFR inhibitor with the PI3K/Akt inhibitor LY294002 led to a significant reduction of ABCG2 expression at low concentrations of the drugs. Based on our results, we assume that EGFR exerts a post-transcriptional enhancing effect on ABCG2 expression via the PI3K/Akt signaling pathway, which can be attenuated by EGFR inhibitors. Blocking the key signaling pathway regulating ABCG2 expression with EGFR inhibitors, combined with the inhibition of ABCG2 with potent modulators might be a promising approach to circumvent MDR

  10. The combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of breast cancer resistance protein (BCRP/ABCG2).

    Kraege, Stefanie; Stefan, Katja; Juvale, Kapil; Ross, Thomas; Willmes, Thomas; Wiese, Michael

    2016-07-19

    During the last decade it has been found that chalcones and quinazolines are promising inhibitors of ABCG2. The combination of these two scaffolds offers a new class of heterocyclic compounds with potentially high inhibitory activity against ABCG2. For this purpose we investigated 22 different heterodimeric derivatives. In this series only methoxy groups were used as substituents as these had been proven superior for inhibitory activity of chalcones. All compounds were tested for their inhibitory activity, specificity and cytotoxicity. The most potent ABCG2 inhibitor in this series showed an IC50 value of 0.19 μM. It possesses low cytotoxicity (GI50 = 93 μM), the ability to reverse MDR and is nearly selective toward ABCG2. Most compounds containing dimethoxy groups showed slight activity against ABCB1 too. Among these three compounds (17, 19 and 24) showed even higher activity toward ABCB1 than ABCG2. All inhibitors were further screened for their effect on basal ATPase activity. Although the basal ATPase activity was partially stimulated, the compounds were not transported by ABCG2. Thus, quinazoline-chalcones are a new class of effective ABCG2 inhibitors. PMID:27100033

  11. The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

    Mahnke, Hanna; Ballent, Mariana; Baumann, Sven; Imperiale, Fernanda; von Bergen, Martin; Lanusse, Carlos; Lifschitz, Adrian L; Honscha, Walther; Halwachs, Sandra

    2016-05-01

    In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk. PMID:26956640

  12. Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

    Ozvegy-Laczka, Csilla; Laczkó, Rozália; Hegedus, Csilla;

    2008-01-01

    Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5......D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the...

  13. Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design

    Ishikawa, Toshihisa; Tamura, Ai; Saito, Hikaru; Wakabayashi, Kanako; Nakagawa, Hiroshi

    2005-10-01

    In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

  14. The human breast cancer resistance protein (BCRP/ABCG2) shows conformational changes with mitoxantrone.

    Rosenberg, Mark F; Bikadi, Zsolt; Chan, Janice; Liu, Xiaoping; Ni, Zhanglin; Cai, Xiaokun; Ford, Robert C; Mao, Qingcheng

    2010-03-14

    BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p12(1) symmetry and projection maps were determined at 5 A resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 A, b = 81.4 A, gamma = 89.8 degrees , and a = 57.3 A, b = 88.0 A, gamma = 89.7 degrees , respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane alpha helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding. PMID:20399185

  15. ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling

    Marriott, Shennea; Baskir, Rubin S.; Gaskill, Christa; Menon, Swapna; Carrier, Erica J.; Williams, Janice; Talati, Megha; Helm, Karen; Alford, Catherine E.; Kropski, Jonathan A.; Loyd, James; Wheeler, Lisa; Johnson, Joyce; Austin, Eric; Nozik-Grayck, Eva

    2014-01-01

    Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2pos mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control...

  16. OCT-1, ABCB1, and ABCG2 Expression in Imatinib-Resistant Chronic Myeloid Leukemia Treated with Dasatinib or Nilotinib

    Kim, Yeo-Kyeoung; Lee, Seung-Shin; Jeong, Sung-Hoon; Ahn, Jae-Sook; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Myung-Geun; Kim, Hyeoung-Joon

    2014-01-01

    This study explored drug transporter expression levels and their impact on clinical response to imatinib and second-generation tyrosine kinase inhibitors (TKIs) in imatinib- resistant chronic myeloid leukemia (CML). Imatinib-resistant chronic phase CML patients treated with dasatinib (n=10) and nilotinib (n=12) were enrolled. The mRNA expression of the OCT-1, ABCG2, and ABCB1 genes was quantified by using paired bone marrow samples obtained before administering imatinib and at the point of de...

  17. The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.

    Macalou, S; Robey, R W; Jabor Gozzi, G; Shukla, S; Grosjean, I; Hegedus, T; Ambudkar, S V; Bates, S E; Di Pietro, A

    2016-05-01

    The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux. PMID:26708291

  18. Evidence for dual mode of action of a thiosemicarbazone, NSC73306: A potent substrate of the multidrug resistance-linked ABCG2 transporter

    Wu, Chung-Pu; Shukla, Suneet; Calcagno, Anna Maria; Hall, Matthew D.; Gottesman, Michael M.; Ambudkar, Suresh V.

    2008-01-01

    Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette transporters, including ABCB1 (P-glycoprotein), ABCG2 and several ABCC family members (MRPs). We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein and it re-sensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4 and MRP5. Our findings demonstrated that NSC73306 is not more toxic to cells that overexpress these transporters compared to their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140–150 nM concentration required for 50% stimulation) and by inhibition of [125I]-Iodoarylazidoprazosin photolabeling (50% inhibition at 250–400 nM) of the substrate-binding site(s). Overall, NSC73306 appears to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4 or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein-overexpressing cells, and by acting as a potent modulator that re-sensitizes ABCG2-expressing cancer cells to chemotherapeutics. PMID:18089722

  19. Association of ABCB1 and ABCG2 single nucleotide polymorphisms with clinical findings and response to chemotherapy treatments in Kurdish patients with breast cancer.

    Ghafouri, Houshiyar; Ghaderi, Bayazid; Amini, Sabrieh; Nikkhoo, Bahram; Abdi, Mohammad; Hoseini, Abdolhakim

    2016-06-01

    The possible interaction between gene polymorphisms and risk of cancer progression is very interesting. Polymorphisms in multi-drug resistance genes have an important role in response to anti-cancer drugs. The present study was aimed to evaluate the possible effects of ABCB1 C3435T and ABCG2 C421A single nucleotide polymorphisms on clinical and pathological outcomes of Kurdish patients with breast cancer. One hundred breast cancer patients and 200 healthy controls were enrolled in this case-control study. Clinical and pathological findings of all individuals were reported, and immunohistochemistry staining was used to assess the tissue expression of specific breast cancer proteins. The ABCB1 C3435T and ABCG2 C421 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The distribution of different genotypes between patient and control groups was only significant for ABCG2 C421A. A allele of ABCG2 C421A polymorphisms were significantly higher in patients than in controls. Patients with AA genotype of ABCG2 C421A were at higher risk of progressing breast cancer. Patients with A allele of ABCG2 had complete response to chemotherapeutic agents. There was no statistically significant association between ABCB1 C3435T and ABCG2 C421A polymorphisms and tissue expression of ER, PR, Her2/neu, and Ki67. The ABCB1 C3435T has no correlation with clinical findings and treatment with chemotherapy drugs. The A allele of ABCG2 C421A may be a risk factor for progression of breast cancer in Kurdish patients. In addition, breast cancer patients with C allele of this polymorphism have weaker response to treatments with anthracyclines and Paclitaxol. PMID:26700668

  20. ABCG2-overexpressing S1-M1-80 cell xenografts in nude mice keep original biochemistry and cell biological properties

    Fang Wang; Yong-Ju Liang; Xing-Ping Wu; Xiao-Dong Su; Li-Wu Fu

    2012-01-01

    S1-M1-80 cells,derived from human colon carcinoma S1 cells,are mitoxantrone-selected ABCG2-overexpressing cells and are widely used in in vitro studies of multidrug resistance (MDR).In this study,S1-M1-80 cell xenografts were established to investigate whether the MDR phenotype and cell biological properties were maintained in vivo.Our results showed that the proliferation,cell cycle,and ABCG2 expression level in S1-M1-80 cells were similar to those in cells isolated from S1-M1-80 cell xenografts (named xS1-M1-80 cells).Consistently,xS1-M1-80 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan,but remained sensitive to the non-ABCG2 substrate cisplatin.Furthermore,the specific ABCG2 inhibitor Ko143 potently sensitized xS1-M1-80 cells to mitoxantrone and topotecan.These results suggest that S1-M1-80 cell xenografts in nude mice retain their original cytological characteristics at 9 weeks.Thus,this model could serve as a good system for further investigation of ABCG2-mediated MDR.

  1. Population Genetic Characteristics Analysis of Exon 9 of ABCG2 Gene in Water Buffalo (Bubalus bubalis)%水牛ABCG2基因第9外显子群体遗传特征分析

    李卓然; 李卫真; 苗永旺; 李大林; 袁跃云; 袁峰; 刘丽仙; 章纯熙

    2012-01-01

    The ATP-binding cassette sub-family G member 2 ( ABCG2) protein plays a crucial role on the milking traits of cattle. But its population genetic characteristics and association with milk produc-tion in buffalo were unclear. In this study, ABCG1 exon 9 of 466 buffalo sampled from 4 river type and 9 swamp type populations were examined using PCR-SSCP and PCR product direct sequencing techniques. The results revealed that sequences of ABCG1 exon 9 in river type and swamp type buffalo were identical, which consist of 251 nucleotides with no SNP discovered, but one T > C substitution (g. SNP44769 T >C) was detected in intron 9. Compared with published data of the bovidae species in GenBank database, one G > A substitution (c. SNP1018 G > A) was found in one river buffalo sequence and lead to the change of the 340th amino acid residues from Valine to Isoleucine (p. V340I) in ABCG2. And the numbers of nucleotide sequence differences among buffalo, cattle, goat and sheep were 5, 8, 11, respectively, which suggest that there are distinct genetic differences between buffalo and the other bovidae species. The results in the present study indicate that ABCG1 exon 9 has been fixed in buffalo and probably had no direct correlation with the trait of buffalo milk yield.%三磷酸腺苷结合转运蛋白G超家族成员2 (ABCG2)对普通奶牛泌乳性状有直接影响.水牛中该基因的遗传特征与产奶性状间是否有遗传关联目前还不十分清楚.本文采用PCR-SSCP和PCR产物直接测序相结合的技术,对4个河流型水牛群体和9个沼泽型水牛群体共计466个个体的ABCG2基因第9外显子进行了变异检测.结果表明,水牛ABCG2基因第9外显子由251个核苷酸组成,所有的河流型和沼泽型水牛序列相同,未发现SNP位点,但在第9内含子内检测到1个T>C替换(g.SNP44769 T>C).与GenBank数据库中已发表的牛科物种同源序列比对后发现,数据库中一条河流型水牛序列该外显子第66

  2. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of

  3. Transporter-Mediated Drug Interaction Strategy for 5-Aminolevulinic Acid (ALA-Based Photodynamic Diagnosis of Malignant Brain Tumor: Molecular Design of ABCG2 Inhibitors

    Toshihisa Ishikawa

    2011-09-01

    Full Text Available Photodynamic diagnosis (PDD is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma. PDD is achieved by a photon-induced physicochemical reaction which is induced by excitation of protoporphyrin IX (PpIX exposed to light. Fluorescence-guided gross-total resection has recently been developed in PDD, where 5-aminolevulinic acid (ALA or its ester is administered as the precursor of PpIX. ALA induces the accumulation of PpIX, a natural photo-sensitizer, in cancer cells. Recent studies provide evidence that adenosine triphosphate (ATP-binding cassette (ABC transporter ABCG2 plays a pivotal role in regulating the cellular accumulation of porphyrins in cancer cells and thereby affects the efficacy of PDD. Protein kinase inhibitors are suggested to potentially enhance the PDD efficacy by blocking ABCG2-mediated porphyrin efflux from cancer cells. It is of great interest to develop potent ABCG2-inhibitors that can be applied to PDD for brain tumor therapy. This review article addresses a pivotal role of human ABC transporter ABCG2 in PDD as well as a new approach of quantitative structure-activity relationship (QSAR analysis to design potent ABCG2-inhibitors.

  4. Gemcitabine upregulates ABCG2/BCRP and modulates the intracellular pharmacokinetic profiles of bioluminescence in pancreatic cancer cells.

    Sun, Yue; Gu, Mancang; Zhu, Lixin; Liu, Junying; Xiong, Yang; Wei, Yinghui; Li, Fanzhu

    2016-03-01

    A lack of methods capable of exploring real-time intracellular drug deposition has since limited the investigation of gemcitabine-induced multidrug resistance in vitro and in vivo. Specifically, resistance induced by D-luciferin, a substrate of the breast cancer resistance protein (ABCG2/BCRP), has recently attracted clinical attention, but further investigation has been limited. Herein, the intracellular pharmacokinetic behavior of D-luciferin was investigated in pancreatic cancer cell lines in real time by using bioluminescence imaging. To achieve this feat, BxPC3 and Panc1 pancreatic cancer cells overexpressing firefly luciferase were treated with gemcitabine in a dose and time gradient manner in vitro. The intracellular pharmacokinetic profiles of each group were then determined through the acquisition of bioluminescent signal intensity of D-luciferin in cells. Simultaneously, key pharmacokinetic parameters including area under the curve, elimination rate constant (K), and mean resident time were calculated according to the noncompartment model. ABCG2 protein levels following gemcitabine treatment were detected through western blot, and gemcitabine showed no significant effect on luciferase activity over dimethyl sulfoxide (DMSO) as a control (P>0.05). However, gemcitabine significantly increased K values while suppressing area under the curve and mean resident time compared with DMSO (Pbioluminescent model and its capability to observe the onset of chemoresistance in real time. PMID:26556627

  5. Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine.

    Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Staud, Frantisek

    2016-01-01

    Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26390406

  6. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    Andersen, Vibeke; Svenningsen, Katrine; Almind Knudsen, Lina; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1....../Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in...

  7. Regulation of ABCG2 expression at the 3' untranslated region of its mRNA through modulation of transcript stability and protein translation by a putative microRNA in the S1 colon cancer cell line

    To, Kenneth K W; Zhan, Zhirong; Litman, Thomas;

    2008-01-01

    ABCG2 is recognized as an important efflux transporter in clinical pharmacology and is potentially important in resistance to chemotherapeutic drugs. To identify epigenetic mechanisms regulating ABCG2 mRNA expression at its 3' untranslated region (3'UTR), we performed 3' rapid amplification of cD...

  8. Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

    Qian, Yan; Wang, Xiao-li; Lv, Zhan-lu; Tysklind, Mats; Guo, Chen; Liang, Bao; Wu, Jia-bing; Yang, Yong-jian; Yang, Yi-shu; Wang, Fei-fei; Duan, Xiao-li; Ma, Jin; Wei, Yong-jie; Wang, Chun-hui; Yang, Li-xin; Zhang, Jin-liang; Shi, Xiao-ming; Wang, Xian-liang

    2016-04-01

    The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747μM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents. PMID:26774982

  9. Ferrocenyl 2,5-Piperazinediones as Tubulin-Binding Organometallic ABCB1 and ABCG2 Inhibitors Active against MDR Cells.

    Wieczorek, Anna; Błauż, Andrzej; Zakrzewski, Janusz; Rychlik, Błażej; Plażuk, Damian

    2016-06-01

    The tubulin-microtubule system is a common target of many anticancer drugs. However, the use of chemotherapeutics frequently leads to the development of a clinically relevant phenomenon of multidrug resistance (MDR). One of the basic mechanisms involved in MDR involves elevated expression and/or activity of several ATP-binding cassette superfamily members (ABC transporters) which are normally responsible for the efflux of xenobiotics or secondary metabolites outside the cell. Here we present the synthesis and biological characteristics of ferrocenyl analogues of plinabulin, i.e. one of the so-called "spindle poisons". We found that replacement of the phenyl group of plinabulin by the ferrocenyl moiety turns this compound into a potent inhibitor of ABCB1 and ABCG2, thus making it possible to overcome the multidrug resistance phenomenon. We also demonstrated that the alkyl group attached to the imidazole moiety of ferrocenyl analogues of plinabulin strongly affects their potency to inhibit tubulin polymerization. PMID:27326336

  10. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

    In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development

  11. Demethylation of miR-9-3 and miR-193a Genes Suppresses Proliferation and Promotes Apoptosis in Non-Small Cell Lung Cancer Cell Lines

    Jinliang Wang

    2013-12-01

    Full Text Available Background: MicroRNAs miR-9-3 and miR-193a have recently been found to be hypermethylated in a variety of non-small cell lung cancer (NSCLC cells and primary human tumors. The objectives of this study were to investigate the role of demethylation of miR-9-3 and miR-193a genes in regulating proliferation and apoptosis in NSCLCs, and to decipher the potential mechanisms underlying the properties. Methods: MTT and population doubling time by flow cytometry were used to assess cell proliferation. Enzyme-Linked Immunosorbent Assay and caspase-3 activity assay were employed to evaluate apoptosis. Real-time RT-PCR and Western blot were used to quantify gene expression at mRNA and protein levels, respectively. Methylation-specific PCR was utilized to assess methylation status. Results: We found that demethylation agent 5-Aza-2'-deoxycytidine (5-AzaC reduced cell numbers and prolonged population doubling time (PDT, and promoted doxorubicin-induced apoptosis in seven NSCLC cell lines with different methylation statuses on miR-9-3 and miR-193a promoter regions: NCI-H1993/NCI-H1915 (miR-9-3+/miR-193a+, NCI-H1975/NCI-H200 (miR-9-3+/miR-193a-, A427/NCI-H2073 (miR-9-3-/miR-193a+, and NCI-H1703 (miR-9-3-/miR-193a-. Treatment with 5-AzaC concomitantly upregulated expression of miR-9-3 and miR-193a, and downregulated their respective target genes NF-κB and Mcl-1. The effects of 5-AzaC were abolished by concomitant knockdown of miR-9-3 and miR-193a using the complex antisense technique, whereas forced ectopic expression of miR-9-3 and miR-193a mimicked the effects of 5-AzaC. We further observed that the strength of proliferation inhibition and apoptosis promotion elicited by 5-AzaC was in the order of NCI-H1993/NCI-H1915 > A427/NCI-H2073 > NCI-H1975/NCI-H200 > NCI-H1703. Conclusions: Methylation-silencing of miR-9-3 and miR-193a may be an important epigenetic mechanisms favoring NSCLC cell growth and survival for carcinogenesis and cancer progression, and

  12. Hypoxia-inducible factor-2a is associated with ABCG2 expression, histology-grade and Ki67 expression in breast invasive ductal carcinoma

    Xiang Lei

    2012-03-01

    Full Text Available Abstract Background Breast cancer is the most common cancer and the leading cause of cancer mortality in women worldwide. Hypoxia is an important factor involved in the progression of solid tumors and has been associated with various indicators of tumor metabolism, angiogenesis and metastasis. But little is known about the contribution of Hypoxia-Inducible Factor-2a (HIF-2a to the drug resistance and the clinicopathological characteristics in breast cancer. Methods Immunohistochemistry was employed on the tissue microarray paraffin sections of surgically removed samples from 196 invasive breast cancer patients with clinicopathological data. The correlations between the expression of HIF-2a and ABCG2 as well as other patients' clinicopathological data were investigated. Results The results showed that HIF-2a was expressed in different intensities and distributions in the tumor cells of the breast invasive ductal carcinoma. A positive staining for HIF-2a was defined as a brown staining observed mainly in the nucleus. A statistically significant correlation was demonstrated between HIF-2a expression and ABCG2 expression (p = 0.001, histology-grade (p = 0.029, and Ki67 (p = 0. 043 respectively. Conclusion HIF-2a was correlated with ABCG2 expression, histology-grade and Ki67 expression in breast invasive ductal carcinoma. HIF-2a could regulate ABCG2 in breast cancer cells, and could be a novel potential bio-marker to predict chemotherapy effectiveness. The hypoxia/HIF-2a/ABCG2 pathway could be a new mechanism of breast cancer multidrug-resistance. Virtual slides http://www.diagnosticpathology.diagnomx.eu/vs/2965948166714795

  13. Expression and Significance of Stem Cell Markers CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 in Pulmonary Squamous Carcinomas

    Xuyong LIN, , , , ,

    2009-04-01

    Full Text Available Background and objective Increasing reports showed that some tumor stem cells were selfrenewal and multi-lineage differentiated in tumors, similar to the normal stem cells in human body. The aim of this study is to observe the expression of stem cell markers in lung squamous carcinoma tissues. Methods Fifty-four lung cancer specimens from surgery were analyzed for CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 expression by using S-P immunohistochemistry. In addition, ten normal lung tissue samples were included as control. Results CK19, Notch3, CD133 and ABCG2 were expressed in 54 Lung cancer tissues, without expression of P75NTR and STRO-1. The expressionrate of CK19, Notch3, CD133 and ABCG2 was 66.67% (36/54, 87.04% (47/54, 50% (27/54, and 61.11% (33/54 respectively. The levels of expression of Notch3, CD133 and ABCG2 were significantly lower in high differentiation group than those in moderate and low differentiation group (P <0.05. The levels of expression of CK19, CD133 and ABCG2 were significantly higher in lymph node metastasis group than those in non-metastasis group (P <0.05. The percentage of total positive cells of four stem cell markers in serial tissue sections was lower than 2%. Conclusion There was expression ofsome stem cell markers in pulmonary squamous carcinomas, and there was relationship between expression degree withdifferentiation degree and lymph node metastasis.

  14. Genistein and Glyceollin Effects on ABCC2 (MRP2 and ABCG2 (BCRP in Caco-2 Cells

    Chandler Schexnayder

    2015-12-01

    Full Text Available The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2 and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6-carboxy-2′,7′-dichloroflourescein (CDF was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.

  15. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind;

    2015-01-01

    transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1....../Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in...... which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that ABCB1 expression identifies a subpopulation of pro-inflammatory Th17 cells which were resistant to treatment with...

  16. Demethylation of the miR-146a promoter by 5-Aza-2’-deoxycytidine correlates with delayed progression of castration-resistant prostate cancer

    Androgen deprivation therapy is the primary strategy for the treatment of advanced prostate cancer; however, after an initial regression, most patients will inevitably develop a fatal androgen-independent tumor. Therefore, understanding the mechanisms of the transition to androgen independence prostate cancer is critical to identify new ways to treat older patients who are ineligible for conventional chemotherapy. The effects of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on the viability and the apoptosis of the androgen-dependent (LNCaP) and androgen-independent (PC3) cell lines were examined by MTS assay and western blot analysis for the activation of caspase-3. The subcutaneous LNCaP xenografts were established in a nude mice model. MiR-146a and DNMTs expressions were analyzed by qRT-PCR and DNA methylation rates of LINE-1 were measured by COBRA-IRS to determine the global DNA methylation levels. The methylation levels of miR-146a promoter region in the different groups were quantified by the bisulfite sequencing PCR (BSP) assay. We validated that 5-Aza-CdR induced cell death and increased miR-146a expression in both LNCaP and PC3 cells. Notably, the expression of miR-146a in LNCaP cells was much higher than in PC3 cells. MiR-146a inhibitor was shown to suppress apoptosis in 5-Aza-CdR-treated cells. In a castrate mouse LNCaP xenograft model, 5-Aza-CdR significantly suppressed the tumors growth and also inhibited prostate cancer progression. Meanwhile, miR-146a expression was significantly enhanced in the tumor xenografts of 5-Aza-CdR-treated mice and the androgen-dependent but not the androgen-independent stage of castrated mice. In particular, the expression of miR-146a was significantly augmented in both stages of the combined treatment (castration and 5-Aza-CdR). Additionally, the methylation percentage of the two CpG sites (−444 bp and −433 bp), which were around the NF-κB binding site at miR-146a promoter, showed the lowest methylation levels among all Cp

  17. The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

    Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V.

    2007-01-01

    Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plu...

  18. Experimental studies on the ABCG2 expression and chemoresistance of suspension sphere-forming PC-3 cells%PC-3悬浮成球细胞中ABCG2表达及其对化学治疗耐药性的实验研究

    张波; 范新兰; 林天歆; 许可慰; 黄海; 黄健

    2012-01-01

    Objective: To explore the ABCG2 expression and the chemoresistance of human prostate cancer sphere-forming PC-3 cells. Methods: PC-3 cells were suspension cultured in vitro with a serum-free medium ( SFM) . The expression of ABCG2 mRNA in sphere-forming PC-3 cells was detected with Real-time quantitative PCR (qRT-PCR) . The sensitivity of sphere-forming PC-3 cells to cisplatin was determined with MTT. Results; The PC-3 sphere-forming cells could survive in SFM and form floating cell spheres. The relative expression quantity of ABCG2 mRNA in the sphere-forming cells was 33. 98-fold higher than that of the PC-3 adherent cells ( P < 0. 05). The IC50 of the sphere-forming cells was 4.26-fold higher than that of the PC-3 adherent cells (P <0.05). Conclusion: The ABCG2 expression of humanprostate cancer sphere-forming PC-3 cells is high, and the chemore-sistance of PC-3 sphere-forming cells is very strong.%目的:探讨人雄激素非依赖性前列腺癌PC-3悬浮成球细胞中ATP结合盒膜转运蛋白G超家族成员2 (ABCG2)的表达及其对化学治疗的耐药性.方法:体外无血清悬浮培养PC-3细胞,逆转录定量-PCR检测PC-3悬浮成球细胞中ABCG2 mRNA的表达,采用MTT法检测PC-3悬浮成球细胞对顺铂的耐药性,计算半数抑制浓度(IC50),并与PC-3贴壁细胞作对比.结果:PC-3悬浮成球细胞可以在无血清培养条件下生存并形成悬浮细胞球,PC-3悬浮成球细胞中ABCG2 mRNA的相对表达水平是PC-3贴壁细胞的33.98倍(P<0.05),其IC50是PC-3贴壁细胞的4.26倍(P<0.05).结论:人PC-3悬浮成球细胞中ABCG2表达水平较高,该类细胞对化学治疗具有较强耐药性.

  19. Expression and Significance of Stem Cell Markers CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 in Pulmonary Squamous Carcinomas

    Xuyong LIN, , , , ,; Liu, Shuli; Liu, Nan; Yang, Xiaoshi; Xu, Hongtao; WANG, ENHUA

    2009-01-01

    Background and objective Increasing reports showed that some tumor stem cells were selfrenewal and multi-lineage differentiated in tumors, similar to the normal stem cells in human body. The aim of this study is to observe the expression of stem cell markers in lung squamous carcinoma tissues. Methods Fifty-four lung cancer specimens from surgery were analyzed for CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 expression by using S-P immunohistochemistry. In addition, ten normal lung tissue sa...

  20. Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells

    The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes

  1. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells

    Robey, R W; Medina-Pérez, W Y; Nishiyama, K;

    2001-01-01

    We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines...... overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100...... microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold...

  2. The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

    Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A; Kawakita, Kazuhito; Takemoto, Daigo

    2016-05-01

    The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans. PMID:27102667

  3. Active DNA demethylation by oxidation and repair

    Zhizhong Gong; Jian-Kang Zhu

    2011-01-01

    DNA methylation and demethylation are increasingly recognized as important epigenetic factors in both plants and animals.DNA methylation,which is catalyzed by DNA methyltransferases (DNMTs),is a relatively stable and heritable modification that controls gene expression,cellular differentiation,genomic imprinting,paramutation,transposon movement,X-inactivation,and embryogenesis [1].The methylation of cytosine to 5-methylcytosine (5mC) is an important example of DNA modification in animals and plants.This highlight concerns DNA demethylation mechanisms in mammals and whether they are similar to that in plants.

  4. 肿瘤干细胞标志物ABCG2、CK19和P63在皮肤日光性角化病、Bowen病及鳞状细胞癌中的表达差异及意义%The differential expression and significance of tumor stem cell markers ABCG2, CK19 and P63 in patients with actinic keratosis, Bowen's Disease and squamous cell carcinoma

    李泓馨; 管海宏; 孙建方; 林麟

    2012-01-01

    Objective To explore the expression and significance of three tumor stem cell markers ABCG2, CK19 and P63 in actinic keratosis (AK), Bowen's disease (BD) and squamous cell carcinoma (SCC). Methods Thirty-seven samples diagnosed as SCC and its precancerous lesion were randomly selected by the Institute of Dermatology of Chinese Academy of Medical Sciences, including 10 AK, 14 BD and 13 SCC. The expression of ABCG2, CK19 and P63 in all these cases was detected by using immunohistochemical MaxVision method. After staining, positively stained ABCG2, CK19 and P63 cases and percentage of positively stained cells in different diseases were statistically analyzed. Results Positively stained substance of ABCG2 and CK19 localized in cell cytoplasm and membrane. The average percentage of ABCG2 and CK19 positively stained cells was significantly higher in SCC (22.1 ± 12.56; 23.5 ± 18.54) than in AK (7.1 ± 6.62; 5.7 ± 4.52) and BD (10.8 ± 9.06; 6.6 ± 4.91) (P0.05). The positive cases of ABCG2 and CK19 increased with the development of pathological extents among AK, BD and SCC, with the ratio of ABCG2 positively stained cases 40%, 71.4%, 84.6% and CK19 positively stained cases 40%, 50%, 92.3%, respectively. Positive stained substance of P63 localized in cell nucleolus. All the 37 cases showed P63 positive, while the number of P63 positively stained cells among the three diseases showed no difference (P>0.05). Conclusion The positive expression level of ABCG2, CK19 and P63 might be related with the development of SCC, and might be potential indicators for the diagnosis of SCC.%目的 检测肿瘤干细胞标志物ABCG2、CK19和P63在皮肤鳞状细胞癌及其癌前病变组织中的表达及意义.方法 选取2010年10月-2011年8月在中国医学科学院皮肤病研究所经病理学确诊的组织学标本37例,其中日光性角化病(AK)10例,Bowen's病(BD) 14例,鳞状细胞癌(scc)13例.采用免疫组织化学MaxVision法检测ABCG2、CK19、P63在各类病变

  5. Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model

    Lu Yang; Huijun Wang; Xin Luo; Pengliang Mao; Weidong Tian; Yujiang Shi; Guoying Huang; Jin Zhang; Duan Ma

    2012-01-01

    DNA methylation and demethylation play important roles in mediating epigenetic regulation.So far,the mechanism of DNA demethylation remains elusive and controversial.Here,we constructed a plasmid,named with pCBS-luc,that contained an artificial CpG island,eight Gal4 DNA-binding domain binding site,an SV40 promoter,and a firefly luciferase reporter gene.The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells.The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained.The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification.The methylation status could be maintained even after 15 passages.The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs.Using our me-pCBS-luc model,we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells.Altogether,we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid,and in this model we found that VP16 could lead to DNA demethylation.We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.

  6. Hedgehog Pathway Inhibitor HhAntag691 Is a Potent Inhibitor of ABCG2/BCRP and ABCB1/Pgp1

    Zhang, Yimao; Laterra, John; Pomper, Martin G.

    2009-01-01

    HhAntag691 (GDC-0449), a low-molecular weight inhibitor of the tumor-promoting hedgehog (Hh) signaling pathway, has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors. Here, we show that HhAntag691 inhibits multiple ATP-binding cassette (ABC) transporters. ATP-binding cassette transporters are within a family of membrane proteins, the overexpression of which is associated with multidrug resistance, a major impediment to s...

  7. Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

    Hu, Xiaoyi V; Rodrigues, Tânia M A; Tao, Haiyan; Baker, Robert K; Miraglia, Loren; Orth, Anthony P; Lyons, Gary E; Schultz, Peter G; Wu, Xu

    2010-08-24

    DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. PMID:20696907

  8. Breast cancer resistance protein (ABCG2) in clinical pharmacokinetics and drug interactions: practical recommendations for clinical victim and perpetrator drug-drug interaction study design.

    Lee, Caroline A; O'Connor, Meeghan A; Ritchie, Tasha K; Galetin, Aleksandra; Cook, Jack A; Ragueneau-Majlessi, Isabelle; Ellens, Harma; Feng, Bo; Taub, Mitchell E; Paine, Mary F; Polli, Joseph W; Ware, Joseph A; Zamek-Gliszczynski, Maciej J

    2015-04-01

    Breast cancer resistance protein (BCRP; ABCG2) limits intestinal absorption of low-permeability substrate drugs and mediates biliary excretion of drugs and metabolites. Based on clinical evidence of BCRP-mediated drug-drug interactions (DDIs) and the c.421C>A functional polymorphism affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of BCRP-mediated DDIs. Although many BCRP substrates and inhibitors have been identified in vitro, clinical translation has been confounded by overlap with other transporters and metabolic enzymes. Regulatory recommendations for BCRP-mediated clinical DDI studies are challenging, as consensus is lacking on the choice of the most robust and specific human BCRP substrates and inhibitors and optimal study design. This review proposes a path forward based on a comprehensive analysis of available data. Oral sulfasalazine (1000 mg, immediate-release tablet) is the best available clinical substrate for intestinal BCRP, oral rosuvastatin (20 mg) for both intestinal and hepatic BCRP, and intravenous rosuvastatin (4 mg) for hepatic BCRP. Oral curcumin (2000 mg) and lapatinib (250 mg) are the best available clinical BCRP inhibitors. To interrogate the worst-case clinical BCRP DDI scenario, study subjects harboring the BCRP c.421C/C reference genotype are recommended. In addition, if sulfasalazine is selected as the substrate, subjects having the rapid acetylator phenotype are recommended. In the case of rosuvastatin, subjects with the organic anion-transporting polypeptide 1B1 c.521T/T genotype are recommended, together with monitoring of rosuvastatin's cholesterol-lowering effect at baseline and DDI phase. A proof-of-concept clinical study is being planned by a collaborative consortium to evaluate the proposed BCRP DDI study design. PMID:25587128

  9. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression.

    Perillo, Bruno; Ombra, Maria Neve; Bertoni, Alessandra; Cuozzo, Concetta; Sacchetti, Silvana; Sasso, Annarita; Chiariotti, Lorenzo; Malorni, Antonio; Abbondanza, Ciro; Avvedimento, Enrico V

    2008-01-11

    Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription. PMID:18187655

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

    Highlights: ► Estrogen signaling and demethylation can both control gene expression in breast cancers. ► Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. ► 137 genes are influenced by both 17β-estradiol and demethylating agent 5-aza-2′-deoxycytidine. ► A set of genes is identified as targets of both estrogen signaling and demethylation. ► 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 17β-estradiol (E2) and a demethylating agent 5-aza-2′-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 these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment

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

    Putnik, Milica, E-mail: milica.putnik@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Zhao, Chunyan, E-mail: chunyan.zhao@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Gustafsson, Jan-Ake, E-mail: jan-ake.gustafsson@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden); Department of Biology and Biochemistry, Science and Engineering Research Center Bldg, University of Houston, Houston, TX 77204-5056 (United States); Dahlman-Wright, Karin, E-mail: karin.dahlman-wright@ki.se [Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge S-14183 (Sweden)

    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. Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

    Visscher, P.T.; Kiene, R.P.; Taylor, B.F.

    1994-01-01

    Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of,intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 ?? mol DMSP l-1 slurry h-1) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 ??mol DMSP l-1 h-1, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 x 107 cells cm-3 sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 x 105 cells cm-3 in the diatom mat (23% cleavers, 77% demethylators), and 9 x 104 cells cm-3 (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.

  13. A New Synthetic Route to Dihydrobenzopyran Via Tandem Demethylation Cyclisation

    Shanmugam, G; S. Shanmuga Sundara Raj; Thirumurugan, R.; N. D. Pradeep Singh; Viswanathan Kasinath; Geetha Gopalakrishnan

    2000-01-01

    A tandem demethylation-cyclisation reaction resulting in the formation of pyran rings using AlCl3/EtSH reagent under mild reaction conditions is reported. X-ray diffraction studies on the intermediate support the suggested mechanism.

  14. Regioselective demethylation of quinoline derivatives. A DFT rationalization

    Belferdi, Fatiha; Merabet, Naima; Belkhiri, Lotfi; Douara, Bachir

    2016-08-01

    Demethylation of compound 2,7-dimethoxyquinoline-3-carbaldehyde 1, is carried out using BBr3. However, all attempts led, either to the starting material or to the regioselective demethylation at position 2 affording the product 4a. The nature (donor or acceptor) and the position of the R (CHO or CN) group is likely to play a role in the preventing the demethylation at position 7. To address this phenomena, the demethylation of 2-chloro-7-methoxyquinoline-3-carbaldehyde 2 and 2,7-dimethoxyquinoline-3-carbaldehyde 3 has been carried out. To support the results obtained, theoretical computations at DFT level (vide infra) have been carried out upon compound 1. The exploration of how the gas-phase demethylation process on Quinoline can be affected at a position 7 center by stepwise substation effects using different electro-donor and attractor groups, show that demethylation process seems to be more favorable when substituent is an electro-donor. This is sustained by bond energy and thermodynamic analyses (vide infra).

  15. Simulated vibrational spectra of aflatoxins and their demethylated products and the estimation of the energies of the demethylation reactions

    Billes, Ferenc; Móricz, Ágnes M.; Tyihák, Ernő; Mikosch, Hans

    2006-06-01

    The structure of four natural mycotoxins, the aflatoxin B 1, B 2, G 1 and G 2 and their demethylated products were optimized with quantum chemical method. The energies and the thermodynamic functions of the molecules were calculated and applied to calculation of the reaction energies of the demethylations. Further results of the calculations are the vibrational force constants, the infrared spectra of the molecules and the assignments of the spectral bands.

  16. ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

    We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of 14C-PhIP (2 μM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 ± 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of 14C-PhIP from maternal to fetal circulation (FM ratio 0.90 ± 0.08 at 6 h, p 14C-PhIP (R = - 0.81, p 14C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells

  17. Demethylation of arsenic limits its volatilization in fungi

    Arsenic (As) biomethylation is increasingly being regarded as a promising method to volatize As from the environment; however, the As volatilization efficiency of most microorganisms is low. Here, the speciation transformation of dimethylarsinic acid (DMA) as an important methylation intermediate in the cells of Fusarium oxysporum CZ-8F1, Penicillium janthinellum SM-12F4, and Trichoderma asperellum SM-12F1 were investigated. These fungal strains have been certified to volatilize As from As-loaded environment. In situ X-ray absorption near edge structure (XANES) indicated that demethylation of DMA with methylarsonic acid (MMA), arsenate [As(V)], and arsenite [As(III)] as intermediates or products occurred in fungal cells after exposure to DMA for 15 days. 36.7–55.7% of the original DMA could lose one or two methyl groups and be changed into MMA or inorganic As. Chromatographic separation of the cell lysates also supported these findings. Thus it comes that demethylation might be a remarkable internal factor limiting As volatilization efficiency. - Highlights: • XAS and chromatographic separation were used to study the speciation change of DMA. • DMA demethylation with MMA, As(V), and As(III) as products occurred in fungal cells. • Demethylation might be responsible for the limited volatilization efficiency of As. - Demethylation might be responsible for the limited methylation efficiency of As

  18. Identification of Three New N-Demethylated and O-Demethyled Bisbenzylisoquinoline Alkaloid Metabolites of Isoliensinine from Dog Hepatic Microsomes

    Su Zeng

    2012-10-01

    Full Text Available Isoliensinine, a natural phenolic bisbenzyltetrahydroisoquinoline alkaloid, has received considerable attention for its potential biological effects such as antioxidant and anti-HIV activities. From the dog hepatic microsomes of isoliensinine, three new N-demethylated and O-demethylated metabolites, 2-N-desmethyl-isoliensinine (M1, 2'-N-desmethylisoliensinine (M2, and 2'-N-6-O-didesmethylisoliensinine (M3, were identified by high-performance liquid chromatography and data-dependent electrospray ionization tandem mass spectrometry. Possible metabolic pathways for isoliensinine have been proposed. The result should prove very helpful for evaluation of the drug-like properties of isoliensinine and other bisbenzylisoquinoline alkaloids.

  19. Anaerobic Mercury Methylation and Demethylation by Geobacter bemidjiensis Bem.

    Lu, Xia; Liu, Yurong; Johs, Alexander; Zhao, Linduo; Wang, Tieshan; Yang, Ziming; Lin, Hui; Elias, Dwayne A; Pierce, Eric M; Liang, Liyuan; Barkay, Tamar; Gu, Baohua

    2016-04-19

    Microbial methylation and demethylation are two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems. Although mercury (Hg) methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjiensis Bem. Here we report, for the first time, that the strain G. bemidjiensis Bem can mediate a suite of Hg transformations, including Hg(II) reduction, Hg(0) oxidation, MeHg production and degradation under anoxic conditions. Results suggest that G. bemidjiensis utilizes a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) as the major reaction product, possibly due to the presence of genes encoding homologues of an organomercurial lyase (MerB) and a mercuric reductase (MerA). In addition, the cells can strongly sorb Hg(II) and MeHg, reduce or oxidize Hg, resulting in both time and concentration-dependent Hg species transformations. Moderate concentrations (10-500 μM) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of Hg methylation and demethylation among anaerobic bacteria, thereby influencing net MeHg production in anoxic water and sediments. PMID:27019098

  20. A New Synthetic Route to Dihydrobenzopyran Via Tandem Demethylation Cyclisation

    G. Shanmugam

    2000-06-01

    Full Text Available A tandem demethylation-cyclisation reaction resulting in the formation of pyran rings using AlCl3/EtSH reagent under mild reaction conditions is reported. X-ray diffraction studies on the intermediate support the suggested mechanism.

  1. Spatial control of protein phosphatase 2A (de)methylation

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2AC) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2AC antibodies revealed a good correlation with the methylation status of PP2AC, demethylated PP2AC being substantially nuclear. Throughout mitosis, demethylated PP2AC is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2AC in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  2. IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

    Highlights: ► DNA methylation is dynamic and flexible and changes rapidly upon cell activation. ► DNA methylation controls the inducible gene expression in a given cell type. ► Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

  3. Cigarette smoke promotes drug resistance and expansion of cancer stem cell-like side population.

    Yi An

    Full Text Available It is well known that many patients continue to smoke cigarettes after being diagnosed with cancer. Although smoking cessation has typically been presumed to possess little therapeutic value for cancer, a growing body of evidence suggests that continued smoking is associated with reduced efficacy of treatment and a higher incidence of recurrence. We therefore investigated the effect of cigarette smoke condensate (CSC on drug resistance in the lung cancer and head and neck cancer cell lines A549 and UMSCC-10B, respectively. Our results showed that CSC significantly increased the cellular efflux of doxorubicin and mitoxantrone. This was accompanied by membrane localization and increased expression of the multi-drug transporter ABCG2. The induced efflux of doxorubicin was reversed upon addition of the specific ABCG2 inhibitor Fumitremorgin C, confirming the role of ABCG2. Treatment with CSC increased the concentration of phosphorylated Akt, while addition of the PI3K inhibitor LY294002 blocked doxorubicin extrusion, suggesting that Akt activation is required for CSC-induced drug efflux. In addition, CSC was found to promote resistance to doxorubicin as determined by MTS assays. This CSC-induced doxurbicin-resistance was mitigated by mecamylamine, a nicotinic acetylcholine receptor inhibitor, suggesting that nicotine is at least partially responsible for the effect of CSC. Lastly, CSC increased the size of the side population (SP, which has been linked to a cancer stem cell-like phenotype. In summary, CSC promotes chemoresistance via Akt-mediated regulation of ABCG2 activity, and may also increase the proportion of cancer stem-like cells, contributing to tumor resilience. These findings underscore the importance of smoking cessation following a diagnosis of cancer, and elucidate the mechanisms of continued smoking that may be detrimental to treatment.

  4. DNA demethylation upregulated Nrf2 expression in Alzheimer's disease cellular model

    Huimin Cao

    2016-01-01

    Full Text Available 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 mRNA and protein levels via down-regulating 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(PH:quinone oxidoreductas (NQO1. Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development.

  5. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine.

    Hackett, Jamie A; Sengupta, Roopsha; Zylicz, Jan J; Murakami, Kazuhiro; Lee, Caroline; Down, Thomas A; Surani, M Azim

    2013-01-25

    Mouse primordial germ cells (PGCs) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5 to E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is an important component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance. PMID:23223451

  6. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ. PMID:26845511

  7. Suppression of gluconeogenic gene expression by LSD1-mediated histone demethylation.

    Dongning Pan

    Full Text Available Aberrant gluconeogenic gene expression is associated with diabetes, glycogen storage disease, and liver cancer. However, little is known how these genes are regulated at the chromatin level. In this study, we investigated in HepG2 cells whether histone demethylation is a potential mechanism. We found that knockdown or pharmacological inhibition of histone demethylase LSD1 causes remarkable transcription activation of two gluconeogenic genes, FBP1 and G6Pase, and consequently leads to increased de novo glucose synthesis and decreased intracellular glycogen content. Mechanistically, LSD1 occupies the promoters of FBP1 and G6Pase, and modulates their H3K4 dimethylation levels. Thus, our work identifies an epigenetic pathway directly governing gluconeogenic gene expression, which might have important implications in metabolic physiology and diseases.

  8. Promotion

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed.

  9. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  10. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers

  11. Pharm GKB: ABCG2 [PharmGKB

    Full Text Available PharmGKB contains no dosing guidelines for this . To report known genotype-based dosing guidelin ... cokinetics Model human liver cell showing blood, bile an d intestinal compartments, indicating tissue speci ... rapeutics. 2015. Wen C C, et al. CA VA Role of the lean ... body mass and of pharmacogenetic variants on the p ...

  12. Demethylation of Circulating Estrogen Receptor Alpha Gene in Cerebral Ischemic Stroke.

    Hsiu-Fen Lin

    Full Text Available Estrogen is involved in neuron plasticity and can promote neuronal survival in stroke. Its actions are mostly exerted via estrogen receptor alpha (ERα. Previous animal studies have shown that ERα is upregulated by DNA demethylation following ischemic injury. This study investigated the methylation levels in the ERα promoter in the peripheral blood of ischemic stroke patients.The study included 201 ischemic stroke patients, and 217 age- and sex-comparable healthy controls. The quantitative methylation level in the 14 CpG sites of the ERα promoter was measured by pyrosequencing in each participant. Multivariate regression model was used to adjust for stroke traditional risk factors. Stroke subtypes and sex-specific analysis were also conducted.The results demonstrated that the stroke cases had a lower ERα methylation level than controls in all 14 CpG sites, and site 13 and site 14 had significant adjusted p-values of 0.035 and 0.026, respectively. Stroke subtypes analysis showed that large-artery atherosclerosis and cardio-embolic subtypes had significantly lower methylation levels than the healthy controls at CpG site 5, site 9, site 12, site 13 and site 14 with adjusted p = 0.039, 0.009, 0.025, 0.046 and 0.027 respectively. However, the methylation level for the patients with small vessel subtype was not significant. We combined the methylation data from the above five sites for further sex-specific analysis. The results showed that the significant association only existed in women (adjusted p = 0.011, but not in men (adjusted p = 0.300.Female stroke cases have lower ERα methylation levels than those in the controls, especially in large-artery and cardio-embolic stroke subtypes. The study implies that women suffering from ischemic stroke of specific subtype may undergo different protective mechanisms to reduce the brain injury.

  13. Demethylation of FANCF gene may be a potential treatment through inhibiting the proliferation of cervical cancer

    Min Li; Chanyu Zhang

    2013-01-01

    Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.

  14. Method for recovering and using lignin in adhesive resins by extracting demethylated lignin

    Schroeder, Herbert A.

    1991-01-01

    Lignin, or a lignin derived material, which has been significantly demethylated (e.g., the demethylated lignin found in the raffinate produced as a by-product of dimethyl sulfide production which can be carried out using the spent liquor from wood pulping operations) can be isolated by a process wherein an organic solvent is added to a lignin-containing aqueous solution. The organic solvent is typically a polar, and at least a partially water-immiscible substance such as, for example, ethyl acetate. The resulting lignin-containing aqueous solution/organic solvent mixture is acidified to produce a water layer which is discarded and an organic solvent layer which contains the demethylated lignin. Upon its recovery, the demethylated lignin is preferably dried and stored until it is used (along with an alkali, an aldehyde and an adhesive filler) in compounding an adhesive of the type generally used in the manufacture of plywood.

  15. DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

    Glover, T W; Coyle-Morris, J; Pearce-Birge, L; Berger, C; Gemmill, R M

    1986-01-01

    Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treate...

  16. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Masao Hattori; Jong-Hyun Lee; Jong-Sik Jin

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−)-arctigenin, the aglycone of arctiin, was demethylated to (−)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarct...

  17. DNA demethylation in normal colon tissue predicts predisposition to multiple cancers.

    Kamiyama, H; Suzuki, K; Maeda, T; Koizumi, K; Miyaki, Y; Okada, S; Kawamura, Y J; Samuelsson, J K; Alonso, S; Konishi, F; Perucho, M

    2012-11-29

    Some colon cancer (CC) patients present synchronous cancers at diagnosis and others develop metachronous neoplasms, but the risk factors are unclear for non-hereditary CC. We showed previously that global DNA demethylation increased with aging and correlated with genomic damage in CC, and we show now that preferentially associates to CCs with wild-type p53. This study aimed to elucidate the extent of DNA hypomethylation in patients with single and multiple CC, its relationship with aging, and its potential as predictive tool. We compared by real-time methylation-specific PCR the relative demethylation level (RDL) of long interspersed nucleotide element-1 (LINE-1) sequences in matched cancer tissues and non-cancerous colonic mucosa (NCM) from patients with single and multiple right-sided CCs. Although no RDL difference was found in NCM from single CC patients and healthy volunteers (P=0.5), there was more demethylation (higher RDL) in NCM from synchronous cancer patients (P=1.1 × 10(-5)) multiple CCs also were more demethylated than single CCs (P=0.0014). High NCM demethylation was predictive for metachronous neoplasms (P=0.003). In multivariate logistic regression analyses RDL was the only independent predictor for metachronous (P=0.02) and multiple (P=4.9 × 10(-5)) tumors. The higher LINE-1 demethylation in NCM from patients with multiple (synchronous and metachronous) tumors (P=9.6 × 10(-7)) was also very significant in patients with tumors without (P=3.8 × 10(-6)), but not with (P=0.16) microsatellite instability. NCM demethylation increased with aging in patients with single tumors, but decreased in those with multiple tumors. Moreover, the demethylation difference between patients with single vs multiple tumors appeared higher in younger (P=3.6 × 10(-4)) than in older (P=0.0016) patients. These results predict that LINE-1 hypomethylation in NCM can be used as an epigenetic predictive biomarker for multiple CC risk. The stronger association of

  18. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: role of jumonji C-domain containing protein 6 in RHA demethylation

    We previously reported that RNA Helicase A (RHA) re-localized from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells, coincident with a reduction in methylation of arginine residues in the RHA C-terminus. To further define the mechanism of RHA demethylation in FMDV-...

  19. The Emerging Nexus of Active DNA Demethylation and Mitochondrial Oxidative Metabolism in Post-Mitotic Neurons

    Huan Meng

    2014-12-01

    Full Text Available The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.

  20. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

  1. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  2. Demethylation of Wheat Straw Alkali Lignin for Application in Phenol Formaldehyde Adhesives

    Yan Song

    2016-05-01

    Full Text Available Lignin is a natural biopolymer with a complex three-dimensional network. It is the second most abundant natural polymer on earth. Commercially, lignin is largely obtained from the waste liquors of pulping and bioethanol productions. In this study, wheat straw alkali lignin (WSAL was demethylated by using an in-situ generated Lewis acid under an optimized demethylation process. The demethylation process was monitored by a semi-quantitative Fourier Transform Infrared Spectroscopy (FTIR method. The demethylated wheat straw alkali lignin (D-WSAL was further characterized by Proton Nuclear Magnetic Resonance (1H NMR, Gel Permeation Chromatography (GPC, and titration methods. After the demethylation process, it was found that the relative value of the methoxy group decreased significantly from 0.82 to 0.17 and the phenolic hydroxyl group increased from 5.2% to 16.0%. Meanwhile, the hydroxyl content increased from 6.6% to 10.3%. GPC results suggested that the weighted averaged molecular weight of D-WSAL was lower than that of WSAL with a smaller polydispersity index. The D-WSAL was then used to replace 60 wt % of phenol to prepare lignin-based phenol formaldehyde adhesives (D-LPF. It was found that both the free formaldehyde content and the free phenol content in D-LPF were less than those of the lignin-based phenol formaldehyde adhesives without lignin demethylation (LPF. Gel time of D-LPF was shortened. Furthermore, the wet and dry bonding strengths of lap shear wood samples bonded using D-LPF were higher than those of the samples bonded using LPF. Therefore, D-WSAL has shown good potential for application in phenol formaldehyde adhesives.

  3. Regeneration and DNA demethylation do not trigger PDX-1 expression in rat hepatocytes

    Rudolf; T; Pillich; Gianfranco; Scarsella; Gianfranco; Risuleo

    2010-01-01

    is in fact able to upregulate Hxk 2 mRNA.We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions.The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1,thus DNA demethylation is not sufficient to override repression of the PDX-1 gene.

  4. Demethylation of host-cell DNA at the site of avian retrovirus integration

    Hejnar, Jiří; Elleder, Daniel; Hájková, P.; Walter, J.; Blažková, Jana; Svoboda, Jan

    2003-01-01

    Roč. 2003, č. 311 (2003), s. 641-648. ISSN 0006-291X Institutional research plan: CEZ:AV0Z5052915 Keywords : DNA methylation and demethylation * integration of retroviruses * gene silencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.836, year: 2003

  5. Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi

    Filley, T.R.; Cody, G.D.; Noser, C. [Carnegie Inst. of Washington, DC (United States). Geophysical Lab.; Goodell, B. [University of Maine, Orono, ME (United States). Dept. of Wood Science; Jellison, J.; Ostrofsky, A. [University of Maine, Orono, ME (United States). Dept. of Biological Sciences

    2002-07-01

    The organic residues produced in the brown-rot (BR) of wood by many basidiomycetes fungi are ubiquitous on most coniferous forest floors. This degraded wood tissue is characterized by low levels of polysaccharides and a high proportion of demethylated lignin with minor glycerol side chain oxidation. Because of the selective enrichment in an aromatic dihydroxy-rich lignin residue, the chemical and biological reactivity of BR degraded wood will be distinctly different from white rot, the other primary class of fungal wood decay, which typically produces oxidized, lignin-depleted residues. The biochemical mechanism by which BR fungi perform this distinctive degradative chemistry is only starting to become known, and molecular studies which examine the chemical changes imparted to lignin over the long-term decay process are lacking. Using {sup 13}C-labeled tetramethylammonium hydroxide thermochemolysis ({sup 13}C -TMAH) and solid state {sup 13}C NMR, we investigated the relationship between lignin oxidation/demethylation and polysaccharide metabolism in a 32-week time series study of spruce sapwood inoculated with either of two BR fungi (Postia placenta and Gloeophyllum trabeum). Our findings demonstrate a close relationship between lignin demethylation and polysaccharide loss and suggest demethylation may play a mechanistic role in polysaccharide loss, possibly by assisting in Fenton reactions where catechol/quinone oxidation and cycling aids in iron reduction. The residue remaining after 16 weeks of decay is devoid of polysaccharides, in contrast to the 68% polysaccharide carbon present in the initial spruce, and exhibits an increased aromatic dihydroxy content (resulting from demethylation of the 3-methoxyl carbon) of up to 22% of the lignin, as determined by {sup 13}C-TMAH thermochemolysis. In a typical soil or porewater environment these chemical changes would make BR residues highly reactive toward redox sensitive polyvalent metals (e.g. ferric iron) and likely

  6. Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

    Benitez-Bribiesca Luis

    2006-08-01

    Full Text Available Abstract Background The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermetylation. Hence, demethylating agents could play a role in overcoming drug resistance. Methods MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. Results MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermetylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. Conclusion We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

  7. Demethylation of Cancer/Testis Antigens and CpG ODN Stimulation Enhance Dendritic Cell and Cytotoxic T Lymphocyte Function in a Mouse Mammary Model

    Jun-Zhong Sun

    2013-01-01

    Full Text Available Background. Cancer/testis antigens (CTAs are ideal targets for cancer immunotherapy in virtue of their restricted expression profile in normal tissues. However, CTA-targeted immunotherapy has been rather disappointing clinical setting for CTAs are downregulated by cytosine-phosphate-guanosine (CpG methylation in their promoter regions, so that tumor cells have low immunogenicity. Methods. We reinduced mouse CTA P1A through demethylation process and generated P1A-specific cytotoxic lymphocytes (CTLs by immunizing BALB/c (H-2d mice with dendritic cells pulsed with a P1A-specific peptide and CpG oligodeoxynucleotide (ODN immune adjuvant. Results. We found that demethylation and CpG ODN immune adjuvant stimulation facilitated DC maturation and enhanced the allogenic capacity of P1A-specific CTLs against target cells both in vitro and in vivo. Conclusions. Our results suggested that CTA induction and immune adjuvant stimulation is a feasible strategy in cancer immunotherapy.

  8. Enhanced binding capability of nuclear factor-κB with demethylated P2X3 receptor gene contributes to cancer pain in rats.

    Zhou, You-Lang; Jiang, Guo-Qin; Wei, Jinrong; Zhang, Hong-Hong; Chen, Wei; Zhu, Hongyan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-10-01

    Nuclear factor-kappa B (NF-κB) signaling is implicated in both cancer development and inflammation processes. However, the roles and mechanisms of NF-κB signaling in the development of cancer-induced pain (CIP) remain unknown. This study was designed to investigate the roles of the p65 subunit of NF-κB in regulation of the purinergic receptor (P2X3R) plasticity in dorsal root ganglion (DRG) of CIP rats. We showed here that tumor cell injection produced mechanical and thermal hyperalgesia, and an enhanced body weight-bearing difference, which was correlated with an upregulation of p65 and P2X3R expression in lumber DRGs and a potentiation of ATP-evoked responses of tibia-innervating DRG neurons. Inhibition of NF-κB signaling using p65 inhibitor pyrrolidine dithiocarbamate, BAY-11-7082, or lentiviral-p65 short-hairpin RNA significantly attenuated CIP and reversed the activities of P2X3R. Interestingly, tumor cell injection led to a significant demethylation of CpG island in p2x3r gene promoter and enhanced ability of p65 to bind the promoter of p2x3r gene. Our findings suggest that upregulation of P2X3R expression was mediated by the enhanced binding capability of p65 with demethylated promoter of p2x3r gene, thus contributing to CIP. NF-κBp65 might be a potential target for treating CIP, a neuropathic pain generated by tumor cell-induced injury to nerves that innervate the skin. PMID:26049406

  9. Demethylation regulation of BDNF gene expression in dorsal root ganglion neurons is implicated in opioid-induced pain hypersensitivity in rats.

    Chao, Yu-Chieh; Xie, Fang; Li, Xueyang; Guo, Ruijuan; Yang, Ning; Zhang, Chen; Shi, Rong; Guan, Yun; Yue, Yun; Wang, Yun

    2016-07-01

    Repeated administration of morphine may result in opioid-induced hypersensitivity (OIH), which involves altered expression of numerous genes, including brain-derived neurotrophic factor (BDNF) in dorsal root ganglion (DRG) neurons. Yet, it remains unclear how BDNF expression is increased in DRG neurons after repeated morphine treatment. DNA methylation is an important mechanism of epigenetic control of gene expression. In the current study, we hypothesized that the demethylation regulation of certain BDNF gene promoters in DRG neurons may contribute to the development of OIH. Real-time RT-PCR was used to assess changes in the mRNA transcription levels of major BDNF exons including exon I, II, IV, VI, as well as total BDNF mRNA in DRGs from rats after repeated morphine administration. The levels of exon IV and total BDNF mRNA were significantly upregulated by repeated morphine administration, as compared to that in saline control group. Further, ELISA array and immunocytochemistry study revealed a robust upregulation of BDNF protein expression in DRG neurons after repeated morphine exposure. Correspondingly, the methylation levels of BDNF exon IV promoter showed a significant downregulation by morphine treatment. Importantly, intrathecal administration of a BDNF antibody, but not control IgG, significantly inhibited mechanical hypersensitivity that developed in rats after repeated morphine treatment. Conversely, intrathecal administration of an inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5-aza-dC) markedly upregulated the BDNF protein expression in DRG neurons and enhanced the mechanical allodynia after repeated morphine exposure. Together, our findings suggest that demethylation regulation of BDNF gene promoter may be implicated in the development of OIH through epigenetic control of BDNF expression in DRG neurons. PMID:26970395

  10. The p16-specific reactivation and inhibition of cell migration through demethylation of CpG islands by engineered transcription factors.

    Zhang, Baozhen; Xiang, Shengyan; Zhong, Qiming; Yin, Yanru; Gu, Liankun; Deng, Dajun

    2012-10-01

    Methylation of CpG islands inactivates transcription of tumor suppressor genes including p16 (CDKN2A). Inhibitors of DNA methylation and histone deacylation are recognized as useful cancer therapeutic chemicals through reactivation of the expression of methylated genes. However, these inhibitors are not target gene-specific, so that they lead to serious side effects as regular cytotoxic chemotherapy agents. To explore the feasibility of methylated gene-specific reactivation by artificial transcription factors, we engineered a set of Sp1-like seven-finger zinc-finger proteins (7ZFPs) targeted to a 21-bp sequence of the p16 promoter and found that these 7ZFPs could bind specifically to the target p16 promoter probe. Then the p16-specific artificial transcription factors (p16ATFs) were made from these 7ZFPs and the transcription activator VP64. Results showed that transient transfection of some p16ATFs selectively up-regulated the endogenous p16 expression in the p16-active 293T cells. Moreover, the transient transfection of the representative p16ATF-6I specifically reactivated p16 expression in the p16-methylated H1299 and AGS cells pretreated with a nontoxic amount of 5'-aza-deoxycytidine (20 and 80 nM, respectively). In addition, stable transfection of the p16ATF induced demethylation of p16 CpG island and trimethylation of histone H3K4, and inhibited recruitment of DNA methyltransferase 1 and trimethylation of H3K9 and H3K27 in the p16 promoter in H1299 cells without 5'-aza-deoxycytidine pretreatment. Notably, inhibition of cell migration and invasion was observed in these p16-reactivated cells induced by transient and stable p16ATF transfection. These results demonstrate that p16ATF not only specifically reactivates p16 expression through demethylation of CpG islands, but also restores methylated p16 function. PMID:22738793

  11. JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1-mediated chromatin response to DNA breaks

    Watanabe, Sugiko; Watanabe, Kenji; Akimov, Vyacheslav;

    2013-01-01

    Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA......-damage response (DDR) pathway. JMJD1C was stabilized by interaction with RNF8, was recruited to DSBs, and was required for local ubiquitylations and recruitment of RAP80-BRCA1 but not 53BP1. JMJD1C bound to RNF8 and MDC1, and demethylated MDC1 at Lys45, thereby promoting MDC1-RNF8 interaction, RNF8-dependent MDC1...... ubiquitylation and recruitment of RAP80-BRCA1 to polyubiquitylated MDC1. Furthermore, JMJD1C restricted formation of RAD51 repair foci, and JMJD1C depletion caused resistance to ionizing radiation and PARP inhibitors, phenotypes relevant to aberrant loss of JMJD1C in subsets of breast carcinomas. These findings...

  12. Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB

    Li, Yuan; Hu, Yanhui; Dong, Congcong; Lu, Hongchao; Zhang, Chang; Hu, Qi; Li, Shifeng; Qin, Heng; Li, Zhong; Wang, Yubang

    2016-01-01

    Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicate that high-doses of DBP and/or MBP exhibit toxicity on testicular function, however, little attention have been paid to the effects of low levels of DBP/MBP on steroidogenesis. As we all know, the steroidogenic acute regulatory protein (StAR) is a key regulator involved in the steroidogenesis. Here we found that, in addition to StAR, MBP/DBP increased the steroidogenesis by a cytoskeletal protein, vimentin. Briefly, in murine adrenocortical tumor (Y1) and the mouse Leydig tumor (MLTC-1) cells, vimentin regulated the secretion of progesterone. When these two cells were exposure to MBP, the DNA demethylation in the vimentin promoter was observed. In addition, MBP also induced the activation of nuclear factor kappa B (NF-κB, a transcriptional regulator of vimentin). These two processes improved the transcriptional elevation of vimentin. Knockdown of NF-κB/vimentin signaling blocked the DBP/MBP-induced steroidogenesis. These in vitro results were also confirmed via an in vivo model. By identifying a mechanism whereby DBP/MBP regulates vimentin, our results expand the understanding of the endocrine disrupting potential of phthalate esters. PMID:26745512

  13. Lipoxygenase—mediated N—demethylation of imipramine and related tricyclic antidepressants

    HuJA; SajaM

    2002-01-01

    The ability of soybean lipoxygenase to mediate the N-demethylation of imipramine and related drugs in the presence of hydrogen peroxide was examined.Under optimal assay conditions,Vmax values of 14 to 18 mol formaldehyde·min-1·mol-1 enzyme were observed.An inhibition of formaldehyde and desipramine formation by nordihydroguaiaretic acid confirmed the lipoxygenase involvement.The blockade of the reaction by glutathione,dithiothreitol butylated hydroxyanisole (BHA) and butylated hydroxytoluene(BHT) indicated the generation of a free radical intermediate from imipramine.Desipramine,trimipramine,clomipramine,and diltiazem,but not amitriptyline and doxepin,were also oxidized,albeit at a lower rate.Collectively,the evidence gathered in this study suggests,for the first time,that tricyclic antidepressant drugs may undergo lipoxygenase-catalyzed N-demethylation.

  14. Characterizing resistance of Erysiphe necator to fungicides belonging to the quinone outside inhibitors and demethylation inhibitors

    Rallos, Lynn Esther Espada

    2013-01-01

    Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under labora...

  15. Dietary compound isoliquiritigenin prevents mammary carcinogenesis by inhibiting breast cancer stem cells through WIF1 demethylation

    Wang, Neng; Wang, Zhiyu; Wang, Yu; Xie, Xiaoming; Shen, Jiangang; Peng, Cheng; You, Jieshu; Peng, Fu; Tang, Hailin; Guan, Xinyuan; Chen, Jianping

    2015-01-01

    Breast cancer stem cells (CSCs) are considered as the root of mammary tumorigenesis. Previous studies have demonstrated that ISL efficiently limited the activities of breast CSCs. However, the cancer prevention activities of ISL and its precise molecular mechanisms remain largely unknown. Here, we report a novel function of ISL as a natural demethylation agent targeting WIF1 to prevent breast cancer. ISL administration suppressed in vivo breast cancer initiation and progression, accompanied b...

  16. Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis

    This work demonstrates that phototaxis stimuli in the archaebacterium Halobacterium halobium control a methylation/demethylation system in vivo through photoactivation of sensory rhodopsin I (SR-I) in either its attractant or repellent signaling form as well as through the repellent receptor sensory rhodopsin II (SR-II, also called phoborhodopsin). The effects of positive stimuli that suppress swimming reversals (i.e., an increase in attractant or decrease in repellent light) and negative stimuli that induce swimming reversals (i.e., a decrease in attractant or increase in repellent light) through each photoreceptor were monitored by assaying release of volatile [3H]methyl groups. This assay has been used to measure [3H]methanol produced during the process of adaptation to chemotactic stimuli in eubacteria. In H. halobium positive photostimuli produce a transient increase in the rate of demethylation followed by a decrease below the unstimulated value, whereas negative photostimuli cause an increase followed by a rate similar to that of the unstimulated value. Photoactivation of the SR-I attractant and simultaneous photoactivation of the SR-II repellent receptors cancel in their effects on demethylation, demonstrating the methylation system is regulated by an integrated signal. Analysis of mutants indicates that the source for the volatile methyl groups is intrinsic membrane proteins distinct from the chromoproteins that share the membrane. A methyl-accepting protein (94 kDa) previously correlated in amount with the SR-I chromoprotein (25 kDa) is shown here to be missing in a recently isolated SR-I-SR-II+ mutant (Flx3b), thus confirming the association of this protein with SR-I. Photoactivated SR-II in mutant Flx3b controls demethylation, predicting the existence of a photomodulated methyl-accepting component distinct from the 94-kDa protein of SR-I

  17. Demethylation of methylmercury in growing rice plants: An evidence of self-detoxification.

    Xu, Xiaohan; Zhao, Jiating; Li, Yunyun; Fan, Yuqin; Zhu, Nali; Gao, Yuxi; Li, Bai; Liu, Hanyu; Li, Yu-Feng

    2016-03-01

    Mercury (Hg) is a global pollutant that poses a serious threat to human and the environment. Rice was found as an important source for human exposure to Hg in some areas. In this study, the transportation and transformation of IHg and MeHg in rice plants exposed to IHg or MeHg were investigated. The IHg and MeHg concentrations in rice roots and shoots collected every five days were analyzed by HPLC-ICP-MS and SR-XANES. When exposed to MeHg, the percent of IHg in rice roots and shoots increased while MeHg decreased significantly, suggesting prominent demethylation of MeHg occurred. However no notable MeHg was found in both roots and shoots of rice plant when exposed to IHg. SR-XANES analysis further confirmed the demethylation of MeHg with rice. This study provides a new finding that demethylation of MeHg could occur in growing rice, which may be a self-defense process of rice plant. PMID:26708765

  18. The role of active DNA demethylation and Tet enzyme function in memory formation and cocaine action.

    Alaghband, Yasaman; Bredy, Timothy W; Wood, Marcelo A

    2016-06-20

    Active DNA modification is a major epigenetic mechanism that regulates gene expression in an experience-dependent manner, which is thought to establish stable changes in neuronal function and behavior. Recent discoveries regarding the Ten eleven translocation (Tet1-3) family of DNA hydroxylases have provided a new avenue for the study of active DNA demethylation, and may thus help to advance our understanding of how dynamic DNA modifications lead to long-lasting changes in brain regions underlying learning and memory, as well as drug-seeking and propensity for relapse following abstinence. Drug addiction is a complex, relapsing disorder in which compulsive drug-seeking behavior can persist despite aversive consequences. Therefore, understanding the molecular mechanisms that underlie the onset and persistence of drug addiction, as well as the pronounced propensity for relapse observed in addicts, is necessary for the development of selective treatments and therapies. In this mini-review, we provide an overview of the involvement of active DNA demethylation with an emphasis on the Tet family of enzymes and 5-hydroxymethylcytosine (5-hmC) in learning and memory, as well as in drug-seeking behavior. Memory and addiction share overlapping molecular, cellular, and circuit functions allowing research in one area to inform the other. Current discrepancies and directions for future studies focusing on the dynamic interplay between DNA methylation and demethylation, and how they orchestrate gene expression required for neuronal plasticity underlying memory formation, are discussed. PMID:26806038

  19. Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

    LING, SHIZHANG; RETTIG, ELENI M.; TAN, MARIETTA; CHANG, XIAOFEI; WANG, ZHIMING; BRAIT, MARIANA; BISHOP, JUSTIN A.; FERTIG, ELANA J.; CONSIDINE, MICHAEL; WICK, MICHAEL J.; HA, PATRICK K.

    2016-01-01

    Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the

  20. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3

    Cloos, Paul A C; Christensen, Jesper; Agger, Karl; Maiolica, Alessio; Rappsilber, Juri; Antal, Torben; Hansen, Klaus H; Helin, Kristian

    2006-01-01

    Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and...... 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of...

  1. Genome-wide CpG island methylation and intergenic demethylation propensities vary among different tumor sites.

    Lee, Seung-Tae; Wiemels, Joseph L

    2016-02-18

    The epigenetic landscape of cancer includes both focal hypermethylation and broader hypomethylation in a genome-wide manner. By means of a comprehensive genomic analysis on 6637 tissues of 21 tumor types, we here show that the degrees of overall methylation in CpG island (CGI) and demethylation in intergenic regions, defined as 'backbone', largely vary among different tumors. Depending on tumor type, both CGI methylation and backbone demethylation are often associated with clinical, epidemiological and biological features such as age, sex, smoking history, anatomic location, histological type and grade, stage, molecular subtype and biological pathways. We found connections between CGI methylation and hypermutability, microsatellite instability, IDH1 mutation, 19p gain and polycomb features, and backbone demethylation with chromosomal instability, NSD1 and TP53 mutations, 5q and 19p loss and long repressive domains. These broad epigenetic patterns add a new dimension to our understanding of tumor biology and its clinical implications. PMID:26464434

  2. Production of Hydrolysable Tannin-Like Structures During the Microbial Demethylation of lignin: An Assessment Using13C-Labeled Tetramethylammonium Hydroxide Thermochemolysis.

    Filley, T.; Blanchette, R.; Nierop, K.; Gamblin, D.

    2003-12-01

    Phenolic compounds in soils are important mediators of microbial activity, metal mobility, soil redox, and soil organic matter building processes. Direct tannin input and the microbial decomposition of lignin in litter and soil are important contributors to this pool of phenols. The ability to accurately assess the relative differences in lignin decay (which are initiated by demethylation and side chain oxidation) among synapyl, coniferyl, and p-coumaryl components of detrital lignin requires the ability to determine microbial demethylation within the complex soil residues. Differentiating between hydrolysable tannins and contributions from advanced lignin decay can be problematic for many of the most common molecular techniques such as alkaline CuO oxidation, pyrolysis GC, and tetramethylammonium hydroxide thermochemolysis because of either the masking effects of derivatizing agents, oxidative damage to ortho-phenols or low volatility of lignin monomers. In this study we investigate lignin demethylation and polyhydroxyl-aromatic production in BC and C horizons of sandy forest soils dominated by oak, the A horizon from a red spruce forest, and controlled microbial inoculation studies of woody tissue using in-line 13C-labeled tetramethylammonium hydroxide thermochemolysis. Both white-rot and brown-rot decay resulted in syringyl demethylation, with the latter exhibiting more aggressive demethylation chemistry, while coniferyl monomer demethylation was essentially restricted to brown-rot decay. In a typical brown-rot sequence demethylation of syringyl components occurs more rapidly than coniferyl units within the same tissue and lower molecular weight fragments are likewise more demethylated than lignin monomers containing the full glycerol side chain. Demethylation of both methoxyl groups in the syringyl monomer is evident in soil horizons as well as laboratory inoculations. The latter may suggest demethylation after lignin depolymerization. Low molecular weight

  3. Simultaneous determination of levophencynonate and its metabolite demethyl levophencynonate in human plasma by liquid chromatography tandem mass spectrometry.

    Li, Bo; Qi, Wenyuan; Shi, Aixin; Hu, Xin; Cheng, Gang

    2016-08-01

    A sensitive and convenient high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to determine levophencynonate and demethyl levophencynonate levels in human plasma simultaneously. Chromatographic separation was achieved on a SHIMADZU Shim-Pack XR C8 column and mass spectrometric analysis was performed by an API5000 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 358.4→156.4 and 344.5→144.2 were used to quantify levophencynonate and demethyl levophencynonate, respectively. This analytical method was fully validated with specificity, linearity, lower limit of quantitation (LLOQ), accuracy, precision, stability, matrix effect and recovery. The linearity of this method were developed to be within the concentration ranges of 10-4000pg/mL for levophencynonate and 25-8000pg/mL for demethyl levophencynonate in human plasma. This method was used in a clinical study which was administrated with single oral dose for Chinese healthy subjects to investigate the pharmacokinetics of levophencynonate and demethyl levophencynonate. PMID:27304783

  4. Evidence that active demethylation mechanisms maintain the genome of carcinoma in situ cells hypomethylated in the adult testis

    Kristensen, D G; Nielsen, J E; Jørgensen, Anne;

    2014-01-01

    absent. Both maintenance and de novo methyltransferases were detected in CIS cells.Conclusion:The data are consistent with the presence of an active DNA de-methylation pathway in CIS cells. The hypomethylated genome of CIS cells may contribute to phenotypic plasticity and invasive capabilities of this...

  5. Design and synthesis of novel 4'-demethyl-4-deoxypodophyllotoxin derivatives as potential anticancer agents.

    Zhu, Xiong; Fu, Junjie; Tang, Yan; Gao, Yuan; Zhang, Shijin; Guo, Qinglong

    2016-02-15

    A group of podophyllotoxin (PPT) derivatives (7a-j) were synthesized by conjugating aryloxyacetanilide moieties to the 4'-hydroxyl of 4'-demethyl-4-deoxypodophyllotoxin (DDPT), and their anticancer activity was evaluated. It was found that the most potent compound 7d inhibited the proliferation of three cancer cell lines with sub to low micromolar IC50 values. Furthermore, it was demonstrated that 7d induced cell cycle arrest in G2/M phase in MGC-803 cells, and regulated the expression of cell cycle check point proteins, such as cyclin A, cyclin B, CDK1, cdc25c, and p21. Finally, 4 mg/kg of 7d reduced the weights and volumes of HepG2 xenografts in mice. Our findings suggest that 7d might be a potential anticancer agent. PMID:26804229

  6. SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells.

    Moriyama, Taishi; Fujimitsu, Yuka; Yoshikai, Yushi; Sasano, Takashi; Yamada, Koji; Murakami, Masataka; Urano, Takeshi; Sugasawa, Kaoru; Saitoh, Hisato

    2014-05-01

    Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG-RNF4 interaction. PMID:24727457

  7. MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis.

    Christ, Bastien; Schelbert, Silvia; Aubry, Sylvain; Süssenbacher, Iris; Müller, Thomas; Kräutler, Bernhard; Hörtensteiner, Stefan

    2012-02-01

    During leaf senescence, chlorophyll (Chl) is broken down to nonfluorescent chlorophyll catabolites (NCCs). These arise from intermediary fluorescent chlorophyll catabolites (FCCs) by an acid-catalyzed isomerization inside the vacuole. The chemical structures of NCCs from Arabidopsis (Arabidopsis thaliana) indicate the presence of an enzyme activity that demethylates the C13(2)-carboxymethyl group present at the isocyclic ring of Chl. Here, we identified this activity as methylesterase family member 16 (MES16; At4g16690). During senescence, mes16 leaves exhibited a strong ultraviolet-excitable fluorescence, which resulted from large amounts of different FCCs accumulating in the mutants. As confirmed by mass spectrometry, these FCCs had an intact carboxymethyl group, which slowed down their isomerization to respective NCCs. Like a homologous protein cloned from radish (Raphanus sativus) and named pheophorbidase, MES16 catalyzed the demethylation of pheophorbide, an early intermediate of Chl breakdown, in vitro, but MES16 also demethylated an FCC. To determine the in vivo substrate of MES16, we analyzed pheophorbide a oxygenase1 (pao1), which is deficient in pheophorbide catabolism and accumulates pheophorbide in the chloroplast, and a mes16pao1 double mutant. In the pao1 background, we additionally mistargeted MES16 to the chloroplast. Normally, MES16 localizes to the cytosol, as shown by analysis of a MES16-green fluorescent protein fusion. Analysis of the accumulating pigments in these lines revealed that pheophorbide is only accessible for demethylation when MES16 is targeted to the chloroplast. Together, these data demonstrate that MES16 is an integral component of Chl breakdown in Arabidopsis and specifically demethylates Chl catabolites at the level of FCCs in the cytosol. PMID:22147518

  8. Suppression of Wnt signaling by the miR-29 family is mediated by demethylation of WIF-1 in non-small-cell lung cancer

    Tan, Min [Department of Respiratory Medicine, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072 (China); Wu, Junjie, E-mail: wujunjiesh@126.com [Department of Pneumology, Changhai Hospital of Shanghai, Second Military Medical University, Shanghai 200433 (China); State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai 200433 (China); Cai, Yong, E-mail: dryongcai@126.com [Department of Radiation Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433 (China)

    2013-09-06

    Highlights: •Dnmt3A and Dnmt3B are involved in the down-regulation of WIF-1 expression in non-small-cell lung cancer. •MiR-29 family members could restore WIF-1 expression through demethylation. •MiR-29s suppress Wnt/β-catenin signaling pathway and inhibit tumor growth. •The expression of miR-29a and miR-29b could be regulated partially in a positive feedback loop. -- Abstract: Wnt inhibitory factor-1 (WIF-1) silencing induced by promoter hypermethylation is a common mechanism of aberrant activation of the Wnt signaling pathway in non-small-cell lung cancer (NSCLC). However, the activity of regulators associated with the methylation of the WIF-1 gene remains unclear. Here, we investigated the role of three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) in the expression of WIF-1. The three DNMTs were up-regulated in NSCLC tumor tissues and suppression of DNMT3A and DNMT3B restored the expression of WIF-1 in NSCLC cells. The miR-29 family (miR-29a, -29b, and -29c), which negatively regulates DNMT3A and DNMT3B, was examined in association with the Wnt/β-catenin signaling pathway. A positive correlation between the expression of WIF-1 and that of MiR-29s was observed in NSCLC tissues. Methylation-specific PCR and Western blotting indicated that miR-29s positively regulate WIF-1 expression by inhibiting the methylation of its promoter. Furthermore, miR-29 overexpression downregulated β-catenin expression, inhibited cell proliferation and induced apoptosis. The expression of miR-29a and miR-29b was partially regulated by DNMT3A and DNMT3B in a positive feedback loop. Taken together, our findings show that miR-29s suppress the Wnt signaling pathway through demethylation of WIF-1 in NSCLC.

  9. Suppression of Wnt signaling by the miR-29 family is mediated by demethylation of WIF-1 in non-small-cell lung cancer

    Highlights: •Dnmt3A and Dnmt3B are involved in the down-regulation of WIF-1 expression in non-small-cell lung cancer. •MiR-29 family members could restore WIF-1 expression through demethylation. •MiR-29s suppress Wnt/β-catenin signaling pathway and inhibit tumor growth. •The expression of miR-29a and miR-29b could be regulated partially in a positive feedback loop. -- Abstract: Wnt inhibitory factor-1 (WIF-1) silencing induced by promoter hypermethylation is a common mechanism of aberrant activation of the Wnt signaling pathway in non-small-cell lung cancer (NSCLC). However, the activity of regulators associated with the methylation of the WIF-1 gene remains unclear. Here, we investigated the role of three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) in the expression of WIF-1. The three DNMTs were up-regulated in NSCLC tumor tissues and suppression of DNMT3A and DNMT3B restored the expression of WIF-1 in NSCLC cells. The miR-29 family (miR-29a, -29b, and -29c), which negatively regulates DNMT3A and DNMT3B, was examined in association with the Wnt/β-catenin signaling pathway. A positive correlation between the expression of WIF-1 and that of MiR-29s was observed in NSCLC tissues. Methylation-specific PCR and Western blotting indicated that miR-29s positively regulate WIF-1 expression by inhibiting the methylation of its promoter. Furthermore, miR-29 overexpression downregulated β-catenin expression, inhibited cell proliferation and induced apoptosis. The expression of miR-29a and miR-29b was partially regulated by DNMT3A and DNMT3B in a positive feedback loop. Taken together, our findings show that miR-29s suppress the Wnt signaling pathway through demethylation of WIF-1 in NSCLC

  10. 5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5 prime CpG island

    Hansen, R.S.; Gartler, S.M. (Univ. of Washington, Seattle (USA))

    1990-06-01

    Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5{prime} CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region.

  11. 5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

    Hansen, R S; Gartler, S M

    1990-01-01

    Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region. Images PMID:1693431

  12. Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysine-specific demethylase 1 (LSD1/KDM1.

    Naoki Sakane

    2011-08-01

    Full Text Available The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51 mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50 mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb. We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1 as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through

  13. N-demethylation of ethylmorphine in pregnant and non-pregnant women and in men: an evaluation of the effects of sex steroids.

    Gerdin, E; Rane, A

    1992-01-01

    1. The effects of oestrogens, testosterone, progesterone and medroxyprogesterone acetate (MPA) on the rate of N-demethylation of ethylmorphine (EM) to norethylmorphine (NEM) were studied in human adult liver microsomes. 2. N-Demethylase activity was found to be inhibited by progesterone and MPA to a similar extent while oestrogens and testosterone had no or negligible effects. 3. These findings prompted us to measure the N-demethylation of EM in relation to serum progesterone concentration in...

  14. Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells

    Zhou, Yang; Hu, Zhengqing

    2016-01-01

    The DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) causes genomic demethylation to regulate gene expression. However, it remains unclear whether 5-aza affects gene expression and cell fate determination of stem cells. In this study, 5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination. PMID:27536218

  15. Determination of parameters influencing methylation and demethylation in tropical lakes in Brazil and Nicaragua

    Increased awareness about the toxicity of mercury (Hg) has during the latest decades resulted in reduced Hg use in industrialised countries. Developing countries, on the contrary, have largely increased their anthropogenic Hg emissions caused by its use in gold mining, transfer of Hg emitting factories from developed countries, and increased burning of coal without appropriate flue gas cleaning. These increased emissions occur mainly in the tropics, where the fate of Hg is not well documented. The aim of the present study is to increase the knowledge about Hg levels and transformations in two tropical areas affected by anthropogenic Hg emissions - the Pantanal wetland in Brazil, housing gold miners using the amalgamation method, and Lake Xolotilan (Managua) in Nicaragua, where a chlor-alkali plant relocated from the USA has emitted much Hg. Actual Hg content in water, biota, and sediment will be determined by atomic fluorescence spectrophotometry and atomic absorption spectrophotometry. Mercury inethylation capacity in sediments and selected biota will be determined with in-situ incubations with 203Hg and subsequent radiological measurements. Factors affecting the methylation and demethylation rates will be identified by varying environmental conditions such as pH, redox potential, conductivity, light, temperature, geochemical factors and population of bacteria. Sediment turnover will be studied by determining fallout cesium (137Cs) in sediment profiles. The study is expected to increase the knowledge about Hg-transformations in the tropics and point out proper measures to reduce health hazards due to Hg-exposure. (author)

  16. Prevention of Helicobacter pylori-induced gastric cancers in gerbils by a DNA demethylating agent.

    Niwa, Tohru; Toyoda, Takeshi; Tsukamoto, Tetsuya; Mori, Akiko; Tatematsu, Masae; Ushijima, Toshikazu

    2013-04-01

    Suppression of aberrant DNA methylation is a novel approach to cancer prevention, but, so far, the efficacy of the strategy has not been evaluated in cancers associated with chronic inflammation. Gastric cancers induced by Helicobacter pylori infection are known to involve aberrant DNA methylation and associated with severe chronic inflammation in their early stages. Here, we aimed to clarify whether suppression of aberrant DNA methylation can prevent H. pylori-induced gastric cancers using a Mongolian gerbil model. Administration of a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), to gerbils (0.125 mg/kg for 50-55 weeks) decreased the incidence of gastric cancers induced by H. pylori infection and N-methyl-N-nitrosourea (MNU) treatment from 55.2% to 23.3% (P testicular atrophy. These results showed that 5-aza-dC treatment can prevent H. pylori-induced gastric cancers and suggested that removal of induced DNA methylation and/or suppression of DNA methylation induction can become a target for prevention of chronic inflammation-associated cancers. PMID:23559452

  17. Regulation of Active DNA Demethylation by a Methyl-CpG-Binding Domain Protein in Arabidopsis thaliana.

    Qi Li

    2015-05-01

    Full Text Available Active DNA demethylation plays crucial roles in the regulation of gene expression in both plants and animals. In Arabidopsis thaliana, active DNA demethylation is initiated by the ROS1 subfamily of 5-methylcytosine-specific DNA glycosylases via a base excision repair mechanism. Recently, IDM1 and IDM2 were shown to be required for the recruitment of ROS1 to some of its target loci. However, the mechanism(s by which IDM1 is targeted to specific genomic loci remains to be determined. Affinity purification of IDM1- and IDM2- associating proteins demonstrated that IDM1 and IDM2 copurify together with two novel components, methyl-CpG-binding domain protein 7 (MBD7 and IDM2-like protein 1 (IDL1. IDL1 encodes an α-crystallin domain protein that shows high sequence similarity with IDM2. MBD7 interacts with IDM2 and IDL1 in vitro and in vivo and they form a protein complex associating with IDM1 in vivo. MBD7 directly binds to the target loci and is required for the H3K18 and H3K23 acetylation in planta. MBD7 dysfunction causes DNA hypermethylation and silencing of reporter genes and a subset of endogenous genes. Our results suggest that a histone acetyltransferase complex functions in active DNA demethylation and in suppression of gene silencing at some loci in Arabidopsis.

  18. Effects of fish CYP inducers on difloxacin N-demethylation in kidney cell of Chinese idle (Ctenopharyngodon idellus).

    Yu, Ling Zhi; Yang, Xian Le; Wang, Xiang Ling; Yu, Wen Juan; Hu, Kun

    2010-09-01

    A drug-drug interaction occurs when the effect of one drug is altered by the presence of another drug which is generally associated with the induction of cytochrome P450s (CYPs) activity. Thus, unexpected treatment failures often happen resulting from inappropriate coadministration in fisheries. However, little information is available about CYP induction in fish. The reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR) belongs to N-demethylation catalyzed mainly by CYP(s). In order to supply useful information on CYP induction, the present study assessed the effects of fish-specific CYP inducers on DIF N-demethylation and enzyme kinetics in kidney cell of Chinese idle (CIK; grass carp (Ctenopharyngodon idellus)) by RP-HPLC. Results demonstrated that the amounts of SAR formation and enzymatic parameters Clint and Vmax were significantly increased due to beta-naphthoflavone (BNF) pretreatment. Therefore, we suggest that CYP1A may be involved in DIF N-demethylation in CIK. This study provides instructive information to ensure treatment success via avoiding CYP induction in fisheries. PMID:19685219

  19. Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism.

    Weber, Alain R; Krawczyk, Claudia; Robertson, Adam B; Kuśnierczyk, Anna; Vågbø, Cathrine B; Schuermann, David; Klungland, Arne; Schär, Primo

    2016-01-01

    Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten-eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET-TDG-BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs. PMID:26932196

  20. DNA去甲基化机制的研究进展%Progress in Study on Mechanism of DNA Demethylation

    曹领改; 张旸; 蓝兴国; 李玉花

    2012-01-01

    As a significant epigenetic modification, DNA methylation plays a critical role in regulating gene expression and maintaining genomic stability in mammals and flowering plants. DNA methylation level and pattern of change will lead to abnormal phenotype or even death. The level and pattern of 5-Mc are determined by both DNA methylation and demethylation processes. Demethylation of DNA can be passive and active. The formation of DNA methylation pattern depends on the active demethylation. Five potential mechanisms of DNA active demethylation are reviewed in this paper: base excision repair involved with DNA glycosylases, base excision repair involved with enzymatic deamination, nucleotide excision repair, oxidative demethylation and hydrolysis.%DNA甲基化作为动植物体内一种重要的表观遗传修饰形式,在调控基因表达、维持基因组的稳定性等方面发挥重要的生物学作用.固有DNA甲基化水平和模式的变化会导致生物的表型异常甚至死亡.而5-甲基胞嘧啶的水平和模式是由DNA甲基化和去甲基化共同决定的.DNA去甲基化可以分为主动去甲基化与被动去甲基化,而基因组甲基化模式的形成主要依赖于主动去甲基化.本文综述了生物体内DNA主动去甲基化五种潜在机制:DNA转葡糖基酶参与的碱基切除修复途径、脱氨酶参与的碱基切除修复途径、核苷酸切除修复途径、氧化作用去甲基化与水解作用去甲基化.

  1. Suppression of Wnt signaling by the miR-29 family is mediated by demethylation of WIF-1 in non-small-cell lung cancer.

    Tan, Min; Wu, Junjie; Cai, Yong

    2013-09-01

    Wnt inhibitory factor-1 (WIF-1) silencing induced by promoter hypermethylation is a common mechanism of aberrant activation of the Wnt signaling pathway in non-small-cell lung cancer (NSCLC). However, the activity of regulators associated with the methylation of the WIF-1 gene remains unclear. Here, we investigated the role of three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) in the expression of WIF-1. The three DNMTs were up-regulated in NSCLC tumor tissues and suppression of DNMT3A and DNMT3B restored the expression of WIF-1 in NSCLC cells. The miR-29 family (miR-29a, -29b, and -29c), which negatively regulates DNMT3A and DNMT3B, was examined in association with the Wnt/β-catenin signaling pathway. A positive correlation between the expression of WIF-1 and that of MiR-29s was observed in NSCLC tissues. Methylation-specific PCR and Western blotting indicated that miR-29s positively regulate WIF-1 expression by inhibiting the methylation of its promoter. Furthermore, miR-29 overexpression downregulated β-catenin expression, inhibited cell proliferation and induced apoptosis. The expression of miR-29a and miR-29b was partially regulated by DNMT3A and DNMT3B in a positive feedback loop. Taken together, our findings show that miR-29s suppress the Wnt signaling pathway through demethylation of WIF-1 in NSCLC. PMID:23939044

  2. Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2.

    Fan, H; Zhang, H; Pascuzzi, P E; Andrisani, O

    2016-02-11

    Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK

  3. Determination of parameters influencing methylation and demethylation in tropical lakes in Brazil and Nicaragua

    Increased awareness about the toxicity of mercury (Hg) has during the latest decades resulted in reduced use of Hg in industrialised countries. Developing countries, on the contrary, have largely increased their anthropogenic Hg emissions caused by its use in gold mining, transfer of Hg emitting factories from developed countries, and increased burning of coal without appropriate flue gas cleaning. The contribution of global Hg sources and the importance of other parameters to increased Hg levels encountered in hydroelectric reservoirs and other areas after flooding is not well understood, especially not in the tropics. The aim of the present study is to increase the knowledge about Hg transformations in tropical areas. Total Hg content in water, biota, and sediment will be determined by atomic absorption and fluorescence spectrophotometry and methyl Hg content in biota by gaschromatography after extraction with acids, hydroxides, and organic solvents. Mercury methylation capacity in sediments, water, and selected biota will be determined with 203Hg and subsequent radiological measurements of insitu incubations. Factors affecting the methylation and demethylation rates will be identified with laboratory incubations with 203Hg at varying environmental conditions such as organic matter, pH, redox potential, conductivity, light, temperature, geochemical factors and populations of bacteria. The populations of bacteria will be determined to quantity by isotope techniques. The first experiments indicate markedly larger methylation capacity as well as bacterial production of incubated samples of Eichhornia crassipes, originating from Brazil, compared to Myriophyllum spicatum from Sweden. The results are the first step to better understand the importance of environmental parameters and bacterial production for methylation of Hg. (author)

  4. Antroquinonol D, isolated from Antrodia camphorata, with DNA demethylation and anticancer potential.

    Wang, Sheng-Chao; Lee, Tzong-Huei; Hsu, Chun-Hua; Chang, Yu-Jia; Chang, Man-Shan; Wang, Yi-Ching; Ho, Yuan-Soon; Wen, Wu-Che; Lin, Ruo-Kai

    2014-06-18

    DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in various human diseases, including cancer. A rational approach to preventing tumorigenesis involves the use of pharmacologic inhibitors of DNA methylation; these inhibitors should reactivate tumor suppressor genes (TSGs) in tumor cells and restore tumor suppressor pathways. Antroquinonol D (3-demethoxyl antroquinonol), a new DNMT1 inhibitor, was isolated from Antrodia camphorata and identified using nuclear magnetic resonance. Antroquinonol D inhibited the growth of MCF7, T47D, and MDA-MB-231 breast cancer cells without harming normal MCF10A and IMR-90 cells. The SRB assay showed that the 50% growth inhibition (GI50) in MCF7, T47D, and MDA-MB-231 breast cancer cells following treatment with antroquinonol D was 8.01, 3.57, and 25.08 μM, respectively. d-Antroquinonol also inhibited the migratory ability of MDA-MB-231 breast cancer cells in wound healing and Transwell assays. In addition, antroquinonol D inhibited DNMT1 activity, as assessed by the DNMT1 methyltransferase activity assay. As the cofactor SAM level increased, the inhibitory effects of d-antroquinonol on DNMT1 gradually decreased. An enzyme activity assay and molecular modeling revealed that antroquinonol D is bound to the catalytic domain of DNMT1 and competes for the same binding pocket in the DNMT1 enzyme as the cofactor SAM, but does not compete for the binding pocket in the DNMT3B enzyme. An Illumina Methylation 450 K array-based assay and real-time PCR assay revealed that antroquinonol D decreased the methylation status and reactivated the expression of multiple TSGs in MDA-MB-231 breast cancer cells. In conclusion, we showed that antroquinonol D induces DNA demethylation and the recovery of multiple tumor suppressor genes, while inhibiting breast cancer growth and migration potential. PMID:24784321

  5. Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell

    Ai-min QIAO; Takashi IKEJIMA; Shini-chi TASHIRO; Satoshi ONODERA; Wei-ge ZHANG; Ying-liang WU

    2006-01-01

    Aim: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. Methods: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. Results: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner.N-demethyl-clarithromycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased.N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. Conclusion: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.

  6. Relationship of the demethylation of the DNA with the induction of the sister chromatid exchanges (SCE) In vivo

    The methylation of the DNA is an epigenetic modification that has an important paper in the regulation of the functionality of the genome of the organisms. It can be altered by demethylation processes, either natural or experimentally induced. The 5-azacytidine (Aza) is a compound that causes the demethylation of the DNA (dm-DNA), inducing with it, expression genic and increase in the frequency of the Sister Chromatid Exchange (SCE). The SCE is a genotoxicity indicator, caused by diverse mutagens and carcinogen. Since the biological meaning and the formation mechanism of this phenomenon has not been totally illustrious, the exploration of the relation between the dm-DNA and the induction of SCE, it could offer new knowledge to explain those queries. The purpose of this work was to study in cells of the mouse bone marrow In vivo, the effect of the Aza on the induction of SCE, based on two aspects: 1) dose answer and 2) the effectiveness of multiple exhibition. To six groups of three to five animals, they are administered Aza to dose of 5, 10, 15 or 20 mg/Kg of weight; in sharp or multiple form, previously to the bromodeoxyuridine supply and 24 h was sacrificed after this; 2 h after an injection with colchicine. Preparations of those metaphases were made, those which were dyed by means of a technique of fluorescence more Giemsa. It was observed that to sharp low dose, the Aza produced an increment in the frequency of SCE that although small it was proportional and statistically significant. To sharp and multiple high doses, the Aza doesn't cause additional increments of SCE, but if toxicity at cellular level and of individuals. It is concluded that a relationship exists between the dm-DNA and the induction of SCE. It is suggested that the total demethylation of the DNA causes 2 SCE/Cell in cells of the mouse bone marrow, or that the cytotoxicity prevents to evidence a bigger induction. (Author)

  7. 牛蒡苷元的脱甲基化研究%Study of Demethylation of ( - )-Arctigenin

    李馥睿; 窦德强; 陈桂荣; 康廷国; 李洪福

    2011-01-01

    Objective: To synthesize demethylic derivatives of ( - )-arctigenin and look for a feasible method of demethylation.Methods: Starting with ( - )-arctigenin, two target products were synthesized with aluminum chloride and hydrobromic acid.Results: Compound 2 was obtained through hydrobromic acid-acetic acid demethylic system.This washigh-yield method which could be operated easily.Conclusion: The optimum technological condition was investigated, refluxing at 110 ℃ for 8h.The yield reached 87%.The structures of the products were determined by 13C-NMR and 1 H-NMR.%目的:以活性天然产物牛蒡苷元为原料,合成其脱甲基衍生物,寻找适宜的脱甲基化方法.方法:以牛蒡苷元为底物,分别以无水氯化铝或氢溴酸为脱甲基试剂进行反应,比较两者脱甲基能力,并制备了目标产物.结果:合成了两个牛蒡苷元脱甲基衍生物,其中化合物2是首次利用氢溴酸-冰醋酸系统进行脱甲基反应而制备得到,采用氢溴酸-冰醋酸系统进行脱甲基反应,收率较高,操作简单.结论:牛蒡苷元脱甲基化最佳工艺条件为:以牛蒡苷元为原料,氢溴酸作为脱甲基试剂,在110℃条件下反应8h,收率87%,并通过核磁共振碳谱和氢谱对目标产物进行了结构确证.

  8. NF-kappaB mediates Gadd45beta expression and DNA demethylation in the hippocampus during fear memory formation.

    Lubin, Farah D.

    2015-01-01

    Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-kB (p65/RelA and cRel) consensus...

  9. NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation

    Jarome, Timothy J.; Butler, Anderson A.; Nichols, Jessica N.; Pacheco, Natasha L.; Lubin, Farah D.

    2015-01-01

    Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus ...

  10. Long-term stability of demethylation after transient exposure to 5-aza-2'-deoxycytidine correlates with sustained RNA polymerase II occupancy.

    Kagey, Jacob D; Kapoor-Vazirani, Priya; McCabe, Michael T; Powell, Doris R; Vertino, Paula M

    2010-07-01

    DNA methyltransferase inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here, we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaCdR) in human breast cancer cells. At the TMS1/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the reengagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single-molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with both unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation, yet a subset of alleles remained stably demethylated for over 3 months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquired H3K9me2 over time, and those alleles that became remethylated retained H3ac. In contrast, CDH1 and ESR1 were remethylated and completely silenced within approximately 1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation. PMID:20587535

  11. Long-term stability of demethylation after transient exposure to 5-aza-2′-deoxycytidine correlates with sustained RNA polymerase II occupancy*

    Kagey, Jacob D.; Kapoor-Vazirani, Priya; McCabe, Michael T.; Powell, Doris R.; Vertino, Paula M.

    2010-01-01

    DNA methyltransferase (DNMT) inhibitors are currently the standard of care for myelodysplastic syndrome and are in clinical trials for leukemias and solid tumors. However, the molecular basis underlying their activity remains poorly understood. Here we studied the induction and long-term stability of gene reactivation at three methylated tumor suppressor loci in response to the DNMT inhibitor 5-aza-2′-deoxycytidine (5-azaCdR)in human breast cancer cells. At the TMS/ASC locus, treatment with 5-azaCdR resulted in partial DNA demethylation, the re-engagement of RNA polymerase II (Pol II), and a shift from a repressive chromatin profile marked with H3K9me2 and H4K20me3 to an active profile enriched in H3ac and H3K4me2. Using a single molecule approach coupling chromatin immunoprecipitation with bisulfite sequencing, we show that H3ac, H3K4me2, and Pol II selectively associated with the demethylated alleles, whereas H3K9me2 preferentially marked alleles resistant to demethylation. H4K20me3 was unaffected by DNA demethylation and associated with unmethylated and methylated alleles. After drug removal, TMS1 underwent partial remethylation yet a subset of alleles remained stably demethylated for over three months. These alleles remained selectively associated with H3K4me2, H3ac, and Pol II and correlated with a sustained low level of gene expression. TMS1 alleles reacquire H3K9me2over time and those alleles that became remethylated retained H3ac. In contrast, CDH1and ESR1 were remethylated and completely silenced within ~1 week of drug removal, and failed to maintain stably unmethylated alleles. Our data suggest that the ability to maintain Pol II occupancy is a critical factor in the long-term stability of drug-induced CpG island demethylation. PMID:20587535

  12. Paroxetine, a cytochrome P450 2D6 inhibitor, diminishes the stereoselective O-demethylation and reduces the hypoalgesic effect of tramadol

    Laugesen, S; Enggaard, T P; Pedersen, R S;

    2005-01-01

    OBJECTIVE: Tramadol hydrochloride (INN, tramadol) exerts its antinociceptive action through a monoaminergic effect mediated by the parent compound and an opioid effect mediated mainly by the O-demethylated metabolite (+)-M1. O-demethylation is catalyzed by cytochrome P450 (CYP) 2D6. Paroxetine is a...... very potent inhibitor of CYP2D6. The objective of this study was to investigate the influence of paroxetine pretreatment on the biotransformation and the hypoalgesic effect of tramadol. METHODS: With and without paroxetine pretreatment (20 mg daily for 3 consecutive days), the formation of M1 and the...

  13. Poly(ADP-ribosylation acts in the DNA demethylation of mouse primordial germ cells also with DNA damage-independent roles.

    Fabio Ciccarone

    Full Text Available Poly(ADP-ribosylation regulates chromatin structure and transcription driving epigenetic events. In particular, Parp1 is able to directly influence DNA methylation patterns controlling transcription and activity of Dnmt1. Here, we show that ADP-ribose polymer levels and Parp1 expression are noticeably high in mouse primordial germ cells (PGCs when the bulk of DNA demethylation occurs during germline epigenetic reprogramming in the embryo. Notably, Parp1 activity is stimulated in PGCs even before its participation in the DNA damage response associated with active DNA demethylation. We demonstrate that PARP inhibition impairs both genome-wide and locus-specific DNA methylation erasure in PGCs. Moreover, we evidence that impairment of PARP activity causes a significant reduction of expression of the gene coding for Tet1 hydroxylases involved in active DNA demethylation. Taken together these results demonstrate new and adjuvant roles of poly(ADP-ribosylation during germline DNA demethylation and suggest its possible more general involvement in genome reprogramming.

  14. Value of the 14C-aminophenazone respiratory test: Inhibition of the demethylation function of the liver by cimetidine

    In 15 patients with healthy liver and peptic ulcer the demethylation capacity of the liver was determined by the 14C-aminophenazone breathing test before and during treatment with the H2 antagonist cimetidine (daily dose 1.0 g) measuring the expired 14CO2 (DPM/mmol CO2/70 kg body weight) before and 1 hour after intake. Influenced by cimetidine the mean 14CO2 value of 724.7 DPM (S.D. 127.7) decreased to 404.1 DPM (S.D. 153.1). The enzyme activities (ALAT, ASAT, AP, AAP, GGT, CHE) and the concentrations of serum bilirubin and serum albumin remained unchanged and did not correlate mutually and with the 14CO2 data. The aminophenazone test proved to be suitable technique for the recognition of disturbances of the MFO system. (author)

  15. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen

    2005-01-01

    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  16. Effects of fish cytochromes P450 inducers and inhibitors on difloxacin N-demethylation in kidney of Chinese idle (Ctenopharyngodon idellus).

    Yu, Ling Zhi; Yang, Xian Le

    2010-05-01

    Cytochromes P450 (CYPs) play key roles in drug metabolism which are widely distributed in kidney in aquatic organisms. CYP(s) mainly catalyzed the N-demethylation reaction of difloxacin (DIF) biotransformation to sarafloxacin (SAR). However, limited information is available about CYP investigation in fish. In order to supply useful information on CYP(s) characterization for DIF N-demethylation, the present study assessed the effects of fish potent CYP inducers and inhibitors on DIF N-demethylation and the inductive and inhibitive enzyme kinetics in kidney of Chinese idle (Ctenopharyngodon idellus) by reversed-phase high-performance liquid chromatography (RP-HPLC). Results demonstrated that the amounts of SAR formation pretreated by β-naphthoflavone (BNF) increased by 1.1-fold and α-naphthoflavone (ANF) inhibited SAR formation level by 0.6-fold at the third day. Enzymatic parameters V(max) and Cl(int) of DIF N-demethylase were increased by 0.56- and 0.38-fold due to β-naphthoflavone (BNF) pretreatment. DIF N-demethylation inhibition by varying ANF concentrations represented a mixed-type inhibition with the value of the inhibition constants (K(i)) 12.9mg/kg. BNF and ANF are the separate typical inducer and inhibitor for CYP1A in fish. Thus, we suggest that CYP1A may be responsible for DIF N-demethylation in kidney. This study provides instructive information to ensure treatment success in fisheries medication with two or more drugs. PMID:21787603

  17. Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump. : Sorafenib inhibits ABCG2 and overcome irinotecan resistance.

    Mazard, Thibault; Causse, Annick; Simony, Joelle; Leconet, Wilhem; Vezzio-Vie, Nadia; Torro, Adeline; Jarlier, Marta; Evrard, Alexandre; Del Rio, Maguy; Assenat, Eric; Martineau, Pierre; Ychou, Marc; Robert, Bruno; Gongora, Celine

    2013-01-01

    Despite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. Therefore, new treatment options are needed to improve survival of patients with irinotecan-refractory CRCs, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemo...

  18. Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation.

    Kondo, Hiroshi; Miura, Takashi; Wada, Kaede C; Takeno, Kiyotoshi

    2007-11-01

    The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria, whose flowering state is stable and the short day plant Pharbitis nil, whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens. On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state. PMID:18251884

  19. Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa Isolates Exhibiting Practical Field Resistance to a Demethylation Inhibitor Fungicide

    Hulvey, Jon; Popko, James T.; Sang, Hyunkyu; Berg, Andrew; Jung, Geunhwa

    2012-01-01

    We investigated genetic factors that govern the reduced propiconazole sensitivity of Sclerotinia homoeocarpa field isolates collected during a 2-year field efficacy study on dollar spot disease of turf in five New England sites. These isolates displayed a >50-fold range of in vitro sensitivity to a sterol demethylation inhibitor (DMI) fungicide, propiconazole, making them ideal for investigations of genetic mechanisms of reduced DMI sensitivity. The CYP51 gene homolog in S. homoeocarpa (ShCYP...

  20. Catalytic Mechanism of the Oxidative Demethylation of 4-(Methoxymethyl)phenol by Vanillyl-Alcohol Oxidase. Evidence for Formation of a p-Quinone Methide Intermediate

    Fraaije, Marco W.; van Berkel, Willem J H

    1997-01-01

    The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied. Using H218O, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of 18O. Enzyme-monitored turnover experiments revealed that for both substrates a process involving flavin reduction is rate determining. During anaer...

  1. Mercury methylation and demethylation by periphyton biofilms and their host in a fluvial wetland of the St. Lawrence River (QC, Canada)

    Wetlands in large rivers are important sites of production of the neurotoxin methylmercury (MeHg), and the periphyton growing on wetland macrophytes are increasingly recognized as key players in this production and transfer in food webs. Information is lacking about mercury methylation (Km) and demethylation (Kd) rates in periphytic biofilms from the Northern Hemisphere, as well as about the drivers of net MeHg production, hampering ecosystem modeling of Hg cycling. Mercury methylation and demethylation rates were measured in periphytic biofilms growing on submerged plants in a shallow fluvial lake located in a temperate cold region (St. Lawrence River, Quebec, Canada). Incubations were performed in situ within macrophyte beds using low-level spikes of 199HgO and Me200Hg stable isotopes as tracers. A direct relationship was observed between Km (0.002 to 0.137 d−1) and [MeHg] in periphyton. A similar relationship was found between Kd (0.096 to 0.334 d−1) and [inorganic Hg]. Periphyton of Lake St. Pierre reached high levels of net MeHg production that were two orders of magnitude higher than those found in local sediment. This production varied through the plant growing season and was mainly driven by environmental variables such as depth of growth, available light, dissolved oxygen, temperature, plant community structure, and productivity of the habitat. - Highlights: • Periphyton Hg methylation and demethylation were studied in a large fluvial lake. • Addition of stable Hg isotopes was used to obtain in situ rates for these processes. • Net methylation was higher in periphyton than in local sediments. • Methylation and demethylation rates fluctuated during the summer. • Key drivers of rates were depth, light, temperature, and community structure

  2. Mercury methylation and demethylation by periphyton biofilms and their host in a fluvial wetland of the St. Lawrence River (QC, Canada)

    Hamelin, Stéphanie; Planas, Dolors [GRIL, Département de sciences biologiques, Université du Québec à Montréal, C.P. 8888, Succursale Centre-Ville, Montreal, Quebec H3C 3P8 (Canada); Amyot, Marc [GRIL, Département de sciences biologiques, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Quebec H3C 3J7 (Canada)

    2015-04-15

    Wetlands in large rivers are important sites of production of the neurotoxin methylmercury (MeHg), and the periphyton growing on wetland macrophytes are increasingly recognized as key players in this production and transfer in food webs. Information is lacking about mercury methylation (K{sub m}) and demethylation (K{sub d}) rates in periphytic biofilms from the Northern Hemisphere, as well as about the drivers of net MeHg production, hampering ecosystem modeling of Hg cycling. Mercury methylation and demethylation rates were measured in periphytic biofilms growing on submerged plants in a shallow fluvial lake located in a temperate cold region (St. Lawrence River, Quebec, Canada). Incubations were performed in situ within macrophyte beds using low-level spikes of {sup 199}HgO and Me{sup 200}Hg stable isotopes as tracers. A direct relationship was observed between K{sub m} (0.002 to 0.137 d{sup −1}) and [MeHg] in periphyton. A similar relationship was found between K{sub d} (0.096 to 0.334 d{sup −1}) and [inorganic Hg]. Periphyton of Lake St. Pierre reached high levels of net MeHg production that were two orders of magnitude higher than those found in local sediment. This production varied through the plant growing season and was mainly driven by environmental variables such as depth of growth, available light, dissolved oxygen, temperature, plant community structure, and productivity of the habitat. - Highlights: • Periphyton Hg methylation and demethylation were studied in a large fluvial lake. • Addition of stable Hg isotopes was used to obtain in situ rates for these processes. • Net methylation was higher in periphyton than in local sediments. • Methylation and demethylation rates fluctuated during the summer. • Key drivers of rates were depth, light, temperature, and community structure.

  3. Validation of 13CO2 breath analysis as a measurement of demethylation of stable isotope labeled aminopyrine in man

    Interval sampling of expired breath as a simple, non-invasive assessment of the effect of liver disease upon hepatic microsomal drug metabolism, has been demonstrated with [14C] dimethylaminoantipyrine (aminopyrine). In order to eliminate radiation risk the authors have validated the use of aminopyrine labeled with the stable, non-radioactive isotope 13C. Simultaneous oral administration of both [14C]- and [13C] aminopyrine to five adult subjects without liver disease as well as five patients with known liver disease, resulted in the excretion of label at nearly identical rates in both individual time collections (r=0.94) as well as cumulative excretion for three hours (r=0.97). An oral dose of 2-mg/kg of [13C) aminopyrine resulted in rates of production of 13CO2 significantly greater than baseline variations in 13CO2 production in the fasting, resting subject. Measurements of a single peak value at one half hour correlated closely with the determination of cumulative appearance over three hours (r=0.96). A consistent reproducible increase in the peak production of 13CO2 was observed when five patients received phenobarbital. Stable isotope labeled aminopyrine may be used to detect the effects of disease and treatment upon hepatic N-demethylation activity in human subjects without incurring any risk from radiation. Furthermore, the availability of another isotopic carbon label should make possible the study of direct drug-drug interaction utilizing CO2 analysis. (Auth.)

  4. Silencing HO-1 sensitizes SKM-1 cells to apoptosis induced by low concentration 5-azacytidine through enhancing p16 demethylation.

    Wang, Ping; Ma, Dan; Wang, Jishi; Fang, Qin; Gao, Rui; Wu, Weibing; Lu, Tangsheng; Cao, Lu

    2015-03-01

    Heme oxygenase-1 was reported previously as a resistance target on acute myelocytic leukemia (AML). We found that HO-1 was resistant to 5-azacytidine (AZA) treatment of myelodysplastic syndrome (MDS), and explored further the relative mechanisms. Patient bone marrow mononuclear cells (n=48) diagnosed as different levels of MDS were collected. Cell growth was evaluated by MTT assay; cell cycle and apoptosis were detected by flow cytometry; mRNA expression was assessed by real-time PCR, protein expression was analyzed through western blotting. Methylation was assessed by MSP. The survival time, and weight of mice were recorded. HO-1 overexpression was observed in SKM-1 cells after AZA treatment comparing to other cell lines. The HO-1 expression in MDS patients with high-risk was higher than in low-risk patients. After HO-1 was silenced by lentivirus-mediated siRNA, the proliferation of SKM-1 cells was effectively inhibited by low concentration AZA, and the cell cycle was arrested in the G0/G1 phase. Upregulation of p16 and changing of p16-relative cell cycle protein was observed after silencing HO-1 in AZA treated SKM-1 cells. In addition, DNMT1 was downregulated following the decrease of HO-1 expression. In vivo, silencing HO-1 inhibited SKM-1 cell growth induced by AZA in a NOD/SCID mouse model. Silencing HO-1 sensitized SKM-1 cells toward AZA, which may be attributed to the influence of HO-1 on AZA-induced p16 demethylation. HO-1 may be one of the targets that enhance the therapeutic effects of AZA on MDS malignant transformation inspiring new treatment methods for high-risk and very high-risk MDS patients in clinical practice. PMID:25585641

  5. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    Yan, Xueying; Ehnert, Sabrina; Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A; Pelisek, Jaroslav; Nussler, Andreas K

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  6. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    Xueying Yan

    Full Text Available The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a as compared to Ad-MSCs isolated from younger donors (<45 a. 5-hydroxymethylcytosine (5 hmC and 5-methylcytonsine (5 mC distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors.

  7. 5-Azacytidine Improves the Osteogenic Differentiation Potential of Aged Human Adipose-Derived Mesenchymal Stem Cells by DNA Demethylation

    Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A.; Pelisek, Jaroslav; Nussler, Andreas K.

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  8. The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals.

    Saito, Masaaki; Suzuki, Koichi; Maeda, Takafumi; Kato, Takaharu; Kamiyama, Hidenori; Koizumi, Kei; Miyaki, Yuichiro; Okada, Shinichiro; Kiyozaki, Hirokazu; Konishi, Fumio

    2012-06-01

    Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers. PMID:22426602

  9. Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes

    Vlková, Veronika; Štěpánek, Ivan; Hrušková, Veronika; Šenigl, Filip; Mayerová, Veronika; Šrámek, Martin; Šímová, Jana; Bieblová, Jana; Indrová, Marie; Hejhal, Tomáš; Dérian, N.; Klatzmann, D.; Six, A.; Reiniš, Milan

    2014-01-01

    Roč. 5, č. 16 (2014), s. 6923-35. ISSN 1949-2553 R&D Projects: GA ČR GAP301/10/2174; GA MZd NT14461 EU Projects: European Commission(XE) 18933 - CLINIGENE Grant ostatní: French state funds within the Investissements d’Avenir program(FR) ANR-11-IDEX-0004-02 Institutional support: RVO:68378050 Keywords : IFNγ signalling pathway * DNA demethylation * tumour Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.359, year: 2014

  10. A Simple, Rapid and Mild One Pot Synthesis of Benzene Ring Acylated and Demethylated Analogues of Harmine under Solvent-free Conditions

    Siddiqui, Bina S.; Syed Imran Hassan; Farhat Farhat; Syed Nawazish Ali; Sabira Begum

    2008-01-01

    A simple, rapid, solvent-free, room temperature one pot synthesis of benzene ring acylated and demethylated analogues of harmine using acyl halides/acid anhydrides and AlCl3 has been developed. Eight different acyl halides/acid anhydrides were used in the synthesis. The resulting mixture of products was separated by column chromatography to afford 10- and 12-monoacyl analogues, along with 10,12-diacyl-11-hydroxy products. In five cases the corresponding 10-acyl-11-hydroxy analogues were also ...

  11. A Simple, Rapid and Mild One Pot Synthesis of Benzene Ring Acylated and Demethylated Analogues of Harmine under Solvent-free Conditions

    Bina S. Siddiqui

    2008-08-01

    Full Text Available A simple, rapid, solvent-free, room temperature one pot synthesis of benzene ring acylated and demethylated analogues of harmine using acyl halides/acid anhydrides and AlCl3 has been developed. Eight different acyl halides/acid anhydrides were used in the synthesis. The resulting mixture of products was separated by column chromatography to afford 10- and 12-monoacyl analogues, along with 10,12-diacyl-11-hydroxy products. In five cases the corresponding 10-acyl-11-hydroxy analogues were also obtained. Yields from the eight syntheses (29 products in total were in the 6-34% range and all compounds were fully characterized.

  12. 5-Methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex

    Zhu, Bing; Zheng, Yong; Hess, Daniel; Angliker, Herbert; Schwarz, Steffen; Siegmann, Michel; Thiry, Stéphane; Jost, Jean-Pierre

    2000-01-01

    We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA ...

  13. Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer

    Christoph, F.; Kempkensteffen, C.; Weikert, S; Köllermann, J.; Krause, H.; Miller, K; Schostak, M; Schrader, M

    2006-01-01

    To examine the significance of the methylation level of the p53 target and tumour suppressor genes apoptotic protease activating factor-1 (APAF-1) and death-associated protein kinase-1 (DAPK-1) in 80 microdissected tumour samples from transitional cell carcinoma (TCC) of the bladder and 80 tumour samples from clear-cell renal cell carcinoma (RCC) as well as from non-tumourous bladder and kidney tissue. Growth-inhibitory effects of the demethylating agents 5-Aza-2′-deoxycytidine (5-Aza-CdR) an...

  14. Cotreatment with Smac mimetics and demethylating agents induces both apoptotic and necroptotic cell death pathways in acute lymphoblastic leukemia cells.

    Gerges, Steve; Rohde, Katharina; Fulda, Simone

    2016-05-28

    Treatment resistance in acute lymphoblastic leukemia (ALL) is often caused by defects in programmed cell death, e.g. by overexpression of Inhibitor of Apoptosis (IAP) proteins. Here, we report that small-molecule Smac mimetics (i.e. BV6, LCL161, birinapant) that neutralize x-linked IAP (XIAP), cellular IAP (cIAP)1 and cIAP2 cooperate with demethylating agents (i.e. 5-azacytidine (5AC) or 5-aza-2'-deoxycytidine (DAC)) to induce cell death in ALL cells. Molecular studies reveal that induction of cell death is preceded by BV6-mediated depletion of cIAP1 protein and involves tumor necrosis factor (TNF)α autocrine/paracrine signaling, since the TNFα-blocking antibody Enbrel significantly reduces BV6/5AC-induced cell death. While BV6/5AC cotreatment induces caspase-3 activation, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) only partly rescues ALL cells from BV6/5AC-induced cell death. This indicates that BV6/5AC cotreatment engages non-apoptotic cell death upon caspase inhibition. Indeed, genetic silencing of key components of necroptosis such as Receptor-Interacting Protein (RIP)3 or mixed lineage kinase domain-like (MLKL) in parallel with administration of zVAD.fmk provides a significantly better protection against BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. Similarly, concomitant administration of pharmacological inhibitors of necroptosis (i.e. necrostatin-1s, GSK'872, dabrafenib, NSA) together with zVAD.fmk is superior in rescuing cells from BV6/5AC-induced cell death compared to the use of zVAD.fmk alone. These findings demonstrate that in ALL cells BV6/5AC-induced cell death is mediated via both apoptotic and necroptotic pathways. Importantly, BV6/5AC cotreatment triggers necroptosis in ALL cells that are resistant to apoptosis due to caspase inhibition. This opens new perspectives to overcome apoptosis resistance with important implications for the development of new treatment strategies

  15. Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1.

    Michael Opel

    Full Text Available In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2, associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9 and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip, we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase

  16. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

    Robey, R W; Honjo, Y; van de Laar, A;

    2001-01-01

    The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compo...

  17. Complex pharmacokinetic behavior of ezetimibe depends on abcc2, abcc3, and abcg2

    D.R. de Waart; M.L.H. Vlaming; C. Kunne; A.H. Schinkel; R.P.J. Oude Elferink

    2009-01-01

    Ezetimibe lowers plasma cholesterol levels by inhibiting the uptake of cholesterol in the intestine. Because of the extensive enterohepatic circulation of ezetimibe, relatively low doses are required to be effective. In blood and bile the majority of ezetimibe is present as a glucuronide conjugate,

  18. In silico prediction of inhibition of promiscuous breast cancer resistance protein (BCRP/ABCG2.

    Yi-Lung Ding

    Full Text Available Breast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart.An in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n= 22, r2 =0.82, qCV2=0.73, RMSE= 0.40, s = 0.24, test set (n =97, q2=0.75-0.89, RMSE= 0.31, s= 0.21, and outlier set (n= 16, q2 =0.72-0.91, RMSE= 0.29, s=0.17. When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity.It was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug-drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance.

  19. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier

    Kumar, Jeyan S.; Bih-Rong Wei; Madigan, James P.; R Mark Simpson; Hall, Matthew D; Gottesman, Michael M.

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the B...

  20. Intestinal ciprofloxacin efflux: the role of breast cancer resistance protein (ABCG2).

    Haslam, I S; Wright, J A; O'Reilly, D A; Sherlock, D J; Coleman, T; Simmons, N L

    2011-12-01

    Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum. PMID:21930826

  1. Folate deprivation induces BCRP (ABCG2) expression and mitoxantrone resistance in Caco-2 cells.

    Lemos, Clara; Kathmann, Ietje; Giovannetti, Elisa; Dekker, Henk; Scheffer, George L; Calhau, Conceição; Jansen, Gerrit; Peters, Godefridus J

    2008-10-01

    Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage. PMID:18623116

  2. The Effects of Lycopene on the Methylation of the GSTP1 Promoter and Global Methylation in Prostatic Cancer Cell Lines PC3 and LNCaP

    Li-Juan Fu

    2014-01-01

    Full Text Available DNA (cytosine-5- methylation silencing of GSTP1 function occurs in prostate adenocarcinoma (PCa. Previous studies have shown that there is an inverse relationship between dietary lycopene intake and the risk of PCa. However, it is unknown whether lycopene reactivates the tumor suppressor gene glutathioneS-transferase-π (GSTP1 by demethylation of the hypermethylated CpGs that act to silence the GSTP1 promoter. Here, we demonstrated that lycopene treatment significantly decreased the methylation levels of the GSTP1 promoter and increased the mRNA and protein levels of GSTP1 in an androgen-independent PC-3 cell line. In contrast, lycopene treatment did not demethylate the GSTP1 promoter or increase GSTP1 expression in the androgen-dependent LNCaP cell line. DNA methyltransferase (DNMT 3A protein levels were downregulated in PC-3 cells following lycopene treatment; however, DNMT1 and DNMT3B levels were unchanged. Furthermore, the long interspersed element (LINE-1 and short interspersed element ALU were not demethylated when treated by lycopene. In LNCaP cells, lycopene treatment did not affect any detected DNMT protein expression, and the methylation levels of LINE-1 and ALU were decreased. These results indicated that the protective effect of lycopene on the prostate is different between androgen-dependent and androgen-independent derived PCa cells. Further, in vivo studies should be conducted to confirm these promising results and to evaluate the potential role of lycopene in the protection of the prostate.

  3. Mercury methylation and demethylation by periphyton biofilms and their host in a fluvial wetland of the St. Lawrence River (QC, Canada).

    Hamelin, Stéphanie; Planas, Dolors; Amyot, Marc

    2015-04-15

    Wetlands in large rivers are important sites of production of the neurotoxin methylmercury (MeHg), and the periphyton growing on wetland macrophytes are increasingly recognized as key players in this production and transfer in food webs. Information is lacking about mercury methylation (Km) and demethylation (Kd) rates in periphytic biofilms from the Northern Hemisphere, as well as about the drivers of net MeHg production, hampering ecosystem modeling of Hg cycling. Mercury methylation and demethylation rates were measured in periphytic biofilms growing on submerged plants in a shallow fluvial lake located in a temperate cold region (St. Lawrence River, Quebec, Canada). Incubations were performed in situ within macrophyte beds using low-level spikes of (199)HgO and Me(200)Hg stable isotopes as tracers. A direct relationship was observed between Km (0.002 to 0.137 d(-1)) and [MeHg] in periphyton. A similar relationship was found between Kd (0.096 to 0.334 d(-1)) and [inorganic Hg]. Periphyton of Lake St. Pierre reached high levels of net MeHg production that were two orders of magnitude higher than those found in local sediment. This production varied through the plant growing season and was mainly driven by environmental variables such as depth of growth, available light, dissolved oxygen, temperature, plant community structure, and productivity of the habitat. PMID:25644842

  4. Metazoan promoters

    Lenhard, Boris; Sandelin, Albin Gustav; Carninci, Piero

    2012-01-01

    Promoters are crucial for gene regulation. They vary greatly in terms of associated regulatory elements, sequence motifs, the choice of transcription start sites and other features. Several technologies that harness next-generation sequencing have enabled recent advances in identifying promoters ...

  5. LSD1 Interacts with Zfp516 to Promote UCP1 Transcription and Brown Fat Program

    Audrey Sambeat; Olga Gulyaeva; Jon Dempersmier; Kevin M. Tharp; Andreas Stahl; Sarah M. Paul; Hei Sook Sul

    2016-01-01

    Zfp516, a brown fat (BAT)-enriched and cold-inducible transcription factor, promotes transcription of UCP1 and other BAT-enriched genes for non-shivering thermogenesis. Here, we identify lysine-specific demethylase 1 (LSD1) as a direct binding partner of Zfp516. We show that, through interaction with Zfp516, LSD1 is recruited to UCP1 and other BAT-enriched genes, such as PGC1α, to function as a coactivator by demethylating H3K9. We also show that LSD1 is induced during brown adipogenesis and ...

  6. Health Promotion

    Povlsen, Lene; Borup, I.

    2015-01-01

    In 1953 when the Nordic School of Public Health was founded, the aim of public health programmes was disease prevention more than health promotion. This was not unusual, since at this time health usually was seen as the opposite of disease and illness. However, with the Ottawa Charter of 1986......, the World Health Organization made a crucial change to view health not as a goal in itself but as the means to a full life. In this way, health promotion became a first priority and fundamental action for the modern society. This insight eventually reached NHV and in 2002 - 50 years after the foundation...... - an associate professorship was established with a focus on health promotion. Nevertheless, the concept of health promotion had been integrated with or mentioned in courses run prior to the new post. Subsequently, a wide spectrum of courses in health promotion was introduced, such as Empowerment for Child...

  7. Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

    Lizano-Soberón Marcela

    2006-12-01

    Full Text Available Abstract Background DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. Methods Cell lines C33A (HPV-, CaSki (HPV-16+ and MS751 (HPV-18+ were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. Results Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. Conclusion These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines

  8. Promoting Models

    Li, Qin; Zhao, Yongxin; Wu, Xiaofeng; Liu, Si

    There can be multitudinous models specifying aspects of the same system. Each model has a bias towards one aspect. These models often override in specific aspects though they have different expressions. A specification written in one model can be refined by introducing additional information from other models. The paper proposes a concept of promoting models which is a methodology to obtain refinements with support from cooperating models. It refines a primary model by integrating the information from a secondary model. The promotion principle is not merely an academic point, but also a reliable and robust engineering technique which can be used to develop software and hardware systems. It can also check the consistency between two specifications from different models. A case of modeling a simple online shopping system with the cooperation of the guarded design model and CSP model illustrates the practicability of the promotion principle.

  9. DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53.

    Alam, S K; Yadav, V K; Bajaj, S; Datta, A; Dutta, S K; Bhattacharyya, M; Bhattacharya, S; Debnath, S; Roy, S; Boardman, L A; Smyrk, T C; Molina, J R; Chakrabarti, S; Chowdhury, S; Mukhopadhyay, D; Roychoudhury, S

    2016-04-01

    Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors. PMID:26494468

  10. Promoting industrialisation

    When the first nuclear power programme is decided upon, automatically the country has to initiate in parallel a programme to modify or add to its current industrial structure and resources. The extent of this new industrialisation depends upon many factors which both, the Government and the Industries have to consider. The Government has a vital role which includes the setting up of the background against which the industrial promotion should take place and in many cases may have also to play an active role all along this programme. Equally, the existing industries have an important role so as to achieve the most efficient participation in the nuclear programme. Invariably the industrial promotional programme will incur a certain degree of transfer of technology, the extent depending on the policies adopted. For this technology transfer to take place efficiently, both the donor and the receiver have to recognise each other's legitimate ambitions and fears. The transfer of technology is a process having a high human content and both donor and receiver have to take this into account. This can be further complicated when there is a difference in culture between them. Technology transfer is carried out within a contractual and organisational framework which will identify the donor (licensor) and the receiver (licensee). This framework may take various forms from a simple cooperative agreement, through a joint-venture organisation right to a standard contract between two separate entities. Each arrangement has its advantages and drawbacks and requires investment of different degrees. One of the keys to a successful industrial promotion is having it carried out in a timely fashion which will be parallel with the nuclear power programme. Experience in some countries has shown the problems when the industrialisation is out of phase with the programme whilst in other cases this industrialisation was at a level and scale unjustified. (author)

  11. Demethylation of oligogalacturonides by FaPE1 in the fruits of the wild strawberry Fragaria vesca triggers metabolic and transcriptional changes associated with defence and development of the fruit

    Osorio, S.; Bombarely, A.; Giavalisco, P; Usadel, B.; Stephens, C.; Araguez, I.; Medina-Escobar, N.; Botella, M.; Fernie, A.; Valpuesta, V.

    2011-01-01

    Ectopic expression of the strawberry (Fragariaxananassa) gene FaPE1 encoding pectin methyl esterase produced in the wild species Fragaria vesca partially demethylated oligogalacturonides (OGAs), which conferred partial resistance of ripe fruits to the fungus Botrytis cinerea. Analyses of metabolic and transcriptional changes in the receptacle of the transgenic fruits revealed channelling of metabolites to aspartate and aromatic amino acids as well as phenolics, flavanones, and sesquiterpenoid...

  12. Breath analysis of 13CO2 following N-demethylation of 13C-aminopyrine: a measure of liver microsomal function

    The hepatic microsomal mixed function oxidase enzyme activity has been measured by N-demethylation of 4-dimethyl-14C-aminopyrine (DAP). Analysis of 14CO2 in expired breath has recently been validated in the rat and man as a measure of this function. In the present study we examine the use of DAP labeled with the stable isotope carbon-13, in order to permit broader clinical application of this test by avoiding radiation exposure. Two mg/kg of 86% enriched 13C-DAP were given orally to 4 normal subjects and 5 patients with cholestatic liver disease. All subjects were fasted overnight and studied at rest. Breath samples were collected at 1/2 hour intervals for 3 hours. In all samples the excess of 13CO2 was significantly greater than the variation in baseline after ingestion of unlabeled DAP. In normal subjects the peak production of 13CO2 occurred in the first 1/2 hour sample. Unlabeled DAP (8 mg/kg) clearance from serum correlated with excess 13CO2 production measured in exhaled breath confirming the 14CO2 results. When phenobarbital (180 mg/day) was administered, an increase in exhaled 13CO2 was observed. Measurement of 13CO2 in breath following DAP provides a reproducible clinical measure of microsomal function and drug induction. The use of stable carbon-13 labeled DAP permits measurement of liver microsomal function in patients who cannot receive radioactive labeled DAP

  13. Effects of the Demethylating Agent, 5-Azacytidine, on Expression of the Kallikrein-Kinin Genes in Carcinoma Cells of the Lung and Pleura

    Joshua Wong

    2011-01-01

    Full Text Available Tissue kallikrein (KLK1 and plasma kallikrein (KLKB1 may regulate the growth and proliferation of tumours of the lung and pleura, through the generation of kinin peptides that signal through the kinin B1 (BDKRB1 and B2 (BDKRB2 receptors. The development and progression of cancer results from genetic mutations, as well as epigenetic changes that include methylation of DNA at CpG islands. The aim of this study was to assess whether expression of the kallikrein-kinin genes in lung cancer and mesothelioma cells is regulated by DNA methylation. Quantitative reverse transcriptase-PCR and immunocytochemistry showed differences in the basal expression of the kallikrein-kinin genes and proteins in lung carcinoma and mesothelioma cells, compared with non-malignant lung epithelial and mesothelial cells, respectively. Following treatment with the demethylating agent, 5-azacytidine (5-AZA, KLKB1 mRNA expression was consistently increased in both lung carcinoma and mesothelioma cells, whereas KLK1, BDKRB1 and BDKRB2 mRNA expression was decreased or unchanged. Increased expression of KLKB1 after 5-AZA treatment suggests it may function as a tumour suppressor gene in cancers of the lung and pleura. Studies on DNA methylation of the kallikrein-kinin genes will enhance understanding of their role in carcinogenesis and provide insights into the importance of kallikreins as tumour biomarkers.

  14. Effects of the Demethylating Agent, 5-Azacytidine, on Expression of the Kallikrein-Kinin Genes in Carcinoma Cells of the Lung and Pleura

    Wong, Joshua; Sia, Yee Yen; Misso, Neil L.; Aggarwal, Shashi; Ng, Angeline; Bhoola, Kanti D.

    2011-01-01

    Tissue kallikrein (KLK1) and plasma kallikrein (KLKB1) may regulate the growth and proliferation of tumours of the lung and pleura, through the generation of kinin peptides that signal through the kinin B1 (BDKRB1) and B2 (BDKRB2) receptors. The development and progression of cancer results from genetic mutations, as well as epigenetic changes that include methylation of DNA at CpG islands. The aim of this study was to assess whether expression of the kallikrein-kinin genes in lung cancer and mesothelioma cells is regulated by DNA methylation. Quantitative reverse transcriptase-PCR and immunocytochemistry showed differences in the basal expression of the kallikrein-kinin genes and proteins in lung carcinoma and mesothelioma cells, compared with non-malignant lung epithelial and mesothelial cells, respectively. Following treatment with the demethylating agent, 5-azacytidine (5-AZA), KLKB1 mRNA expression was consistently increased in both lung carcinoma and mesothelioma cells, whereas KLK1, BDKRB1 and BDKRB2 mRNA expression was decreased or unchanged. Increased expression of KLKB1 after 5-AZA treatment suggests it may function as a tumour suppressor gene in cancers of the lung and pleura. Studies on DNA methylation of the kallikrein-kinin genes will enhance understanding of their role in carcinogenesis and provide insights into the importance of kallikreins as tumour biomarkers. PMID:21904690

  15. Homocysteine harasses the imprinting expression of IGF2 and H19 by demethylation of differentially methylated region between IGF2/H19 genes

    Lijuan Li; Jing Xie; Meng Zhang; Shuren Wang

    2009-01-01

    Homocysteine (Hcy) can induce proliferation of vascular smooth muscle cells (VSMCs), which is a key event in the genesis of the lesions of atherosclerosis. Insulinlike growth factor 2 (IGF2) and 1119 are two important regulating molecules of cell proliferation. The role of Hcy in the proliferation of smooth muscle cell by regulating IGF2 and HI9 has not been shown or analyzed so far. This study aims to investigate the potential impact of Hcy on gene imprinting of IGF2 and 1-119.Cultured human umbilical VSMCs were treated with different concentrations of Hcy. The DNA methylation status of VSMCs was assayed by nested methylationspecific polymerase chain reaction (PCR). The mRNA levels of H19, IGF2, and CCCTC-binding factor (CTCF) were detected by reverse transcription PCR,and the protein expression of IGF2 by Western blotting. The results showed that the Hcy treatment resulted in hypomethylation of the sixth CTCF-binding site upstream of 1119 of VSMCs. The expression of H19 was increased, whereas the IGF2 mRNA and protein were decreased, the CTCF expression increased with the increase in Hcy concentration. These data indicated that Hcy could induce hypomethylation of the sixth CTCF-binding sites upstream of 1119, which is an important regulating area for the imprinting expression of IGF2 and 1119. The increased CTCF expression may be a potential mechanism for the demethylation modification of DNA, which resulted from the Hcy treatment.

  16. Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

    Chih-Jie Shen

    2014-01-01

    Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

  17. Promoter methylation regulates cyclooxygenase expression in breast cancer

    Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract. Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential. We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis). These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype

  18. A novel diterpene skeleton: identification of a highly aromatic, cytotoxic and antioxidant 5-methyl-10-demethyl-abietane-type diterpene from Premna serratifolia.

    Habtemariam, Solomon; Varghese, George K

    2015-01-01

    Premna serratifolia Linn. (syn: . P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.) is a member of the Verbenaceae family that is extensively used in the Ayurvedic system of medicine in India. As part of our continuous pharmacological and phytochemical studies on medicinal plants, we have screened the methanolic extracts of leaves, root bark (RB) and root wood of P. serratifolia for cytotoxic activity against two cancer cell lines: SHSY-5Y neuroblastoma and B16 melanoma cells. The RB extract that showed promising activity was fractionated using solvents of increasing polarity followed by a combination of Sephadex LH-20 column and Combiflash chromatography as well as HPLC to afford the active principle. Comprehensive spectroscopic analysis including 1D and 2D NMR (COSY, HMQC, HMBC, NOESY) and MS analysis revealed the identity of the isolated compound as 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1[10],6,8,11,13-pentene that appears to be a novel compound based on a new diterpene skeleton. The cytotoxic activity of the isolated compound was 21 and 23 times higher than the crude extract against the SHSY-5Y and B16 cells, respectively. The novel compound also possesses in vitro antioxidant effects as evidenced by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging effect where an IC50 value of 20.4 ± 1.3 μM was obtained. In comparison, the positive control, caffeic acid, showed an IC50 value of 14.4 ± 1.6 μM. PMID:25250850

  19. Promoter hyper-methylation of P16 during neoplastic transformation of rat respiratory tract epithelial cells

    To investigate whether p16 hyper-methylation is involved in the silencing of p16 expression and the development of rat lung tumors, p16 status and neoplastic transformation of several respiratory tract epithelial cell lines were examined. Analysis utilizing methylation specific PCR (MSP) method revealed that virus-immortalized SV40T2 cells had un-methylated status and that benzo [a ] pyrene-induced BP cells displayed heterogeneous methylation status of the p6 promoter region. On the other hand, BP130, BP270 and BP(P)Tu cells derive d from BP cells, and gamma ray-transformed RTiv3 cells displayed complete methylation of the gene. The MSP and PCR of genomic DNA in the p16 region did not amplify product in PuD2 cells established from the plutonium-induced lung tumor. Expression analysis of p16 mRNA by RT-PCR demonstrated that SV40T2 and BP cells expressed the p16 transcript. De-methylating agent, 5AzaC de-methylated partially the p16 promoter region of BP(P)Tu and BP cells and increased expression of the p16 transcript. Tumorigenicity assay utilizing inoculation of the cells into nude mouse revealed that SV40T2 and RTiv3 cells had no tumorigenicity. Treatment of BP(P)Tu and BP cells with 5AzaC decreased the cell growth in nude mouse. These results indicate that the hyper-methylation of p16 promoter region occurs at the early stage of neoplastic transformation processes and the gene silencing following the methylation is partially concerned with the tumorigenicity of rat respiratory tract cells. Homozygous deletion and lack of expression of the p16 may also account for the mechanisms of tumorigenicity. (author)

  20. Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

    Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer

  1. Different involvement of promoter methylation in the expression of organic cation/carnitine transporter 2 (OCTN2 in cancer cell lines.

    Qiang Qu

    Full Text Available Organic cation/carnitine transporter 2 (OCTN2 is responsible for the cellular uptake of the antineoplastic agent, oxaliplatin. Epigenetic modification is a possible mechanism of altered drug-transporter expression in cancers, leading to altered efficacy of chemotherapeutic drugs. However, the mechanisms governing OCTN2 regulation are not completely understood. In this study, the low levels of OCTN2 in HepG2 and LS174T cells were elevated by the demethylating reagent, decitabine (DCA. To further reveal the epigenetic mechanism of down-regulation of OCTN2, we found that Region-1 within the OCTN2 promoter (spanning -354 to +85 was a determinant of OCTN2 expression in a luciferase reporter assay. Moreover, methylation-specific PCR (MSP and bisulfite genomic sequencing showed that the degree of individual methylated CpG sites within this region was inversely correlated with the levels of OCTN2 in different cancer cells. Application of DCA to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin. Thus, we identified that promoter methylation is responsible for epigenetic down-regulation of OCTN2 in HepG2 and LS174T cells. Given the essential role of OCTN2 in cancer cell uptake of chemotherapeutics, and thus treatment efficacy, pretreatment with a demethylating reagent is a possible strategy for optimizing pharmacotherapies against cancers.

  2. Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan

    Maishi, Nako; Ohba, Yusuke; Akiyama, Kosuke; Ohga, Noritaka; Hamada, Jun-ichi; Nagao-Kitamoto, Hiroko; Alam, Mohammad Towfik; Yamamoto, Kazuyuki; Kawamoto, Taisuke; Inoue, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2016-01-01

    Tumour blood vessels are gateways for distant metastasis. Recent studies have revealed that tumour endothelial cells (TECs) demonstrate distinct phenotypes from their normal counterparts. We have demonstrated that features of TECs are different depending on tumour malignancy, suggesting that TECs communicate with surrounding tumour cells. However, the contribution of TECs to metastasis has not been elucidated. Here, we show that TECs actively promote tumour metastasis through a bidirectional interaction between tumour cells and TECs. Co-implantation of TECs isolated from highly metastatic tumours accelerated lung metastases of low metastatic tumours. Biglycan, a small leucine-rich repeat proteoglycan secreted from TECs, activated tumour cell migration via nuclear factor-κB and extracellular signal–regulated kinase 1/2. Biglycan expression was upregulated by DNA demethylation in TECs. Collectively, our results demonstrate that TECs are altered in their microenvironment and, in turn, instigate tumour cells to metastasize, which is a novel mechanism for tumour metastasis. PMID:27295191

  3. Determination of total and organic mercury and evaluation of methylation and demethylation processes in sediments of the Rio Grande Reservoir, State of Sao Paulo, Brazil

    The Rio Grande reservoir is located in the metropolitan area of Sao Paulo and it is a very important water supply for this region. In the present study bottom waters and sediment samples collected in this reservoir, in four sampling points, in four campaigns, from September 2008 to January 2010, were analyzed. Firstly total Hg was determined in sediment and bottom waters by cold vapor atomic absorption technique (CV AAS). Following, the analytical methodology for organic Hg was adapted from literature, where the organomercurial compounds were extracted with dichloromethane in acid medium and subsequent destruction of organic compounds by bromine chloride. The validation of this methodology, in terms of precision and accuracy, was performed by means of IAEA 405 and BCR-CRM 580 reference materials analyses. For methylation and demethylation processes evaluation in this environment, the following physical and chemical parameters were assessed, in situ: pH, water temperature, redox potential (EH), transparency and depth. For the sediment samples, granulometry, total organic carbon, sulphate-reducing bacteria, total N and P, besides the metals Co, Cu, Fe and Mn were evaluated. The selection of these parameters was related to the factors that influence the behavior of MeHg in the sediments and its transition zone. Total Hg ranging from 1.0 to 71.0 mg kg'-1 and organic mercury from -1 in sediments and methylation rates from 0.06 to 1.4% were found, along the reservoir. Different methylation conditions along the reservoir and its influences were also discussed. As supplementary study the concentration of some metals and trace elements in the sediments by neutron activation analysis technique was determined. As, Ba, Br, Co, Cr, Cs, Hf, Fe, Na, Rb, Sb, Sc and Zn and rare earth elements Ce, Eu, La, Lu, Nd, Sm and Yb were determined. The enrichment factor in relation to earth crust values using Sc as normalizer element reached values higher than 2.0 for the elements As, Br, Sb

  4. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2)

    Litman, Thomas; Brangi, M; Hudson, E;

    2000-01-01

    Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also...... known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR...... studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required....

  5. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump

    H. Burger (Herman); H. van Tol; A.W.M. Boersma (Anton); M. Brok (Mariël); E.A.C. Wiemer (Erik); G. Stoter (Gerrit); K. Nooter (Kees)

    2004-01-01

    textabstractImatinib mesylate (STI571), a potent tyrosine kinase inhibitor, is successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. However, the intended chronic oral administration of imatinib may lead to development of cellular r

  6. 5-azacytidine reduces methylation, promotes differentiation and induces tumor regression in a patient-derived IDH1 mutant glioma xenograft

    Borodovsky, Alexandra; Salmasi, Vafi; Turcan, Sevin; Fabius, Armida W. M.; Baia, Gilson S.; Eberhart, Charles G.; Weingart, Jon D.; Gallia, Gary L.; Baylin, Stephen B.; Chan, Timothy A.; Riggins, Gregory J.

    2013-01-01

    Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas and are characterized by the production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate by the mutant enzyme. 2-HG is an “oncometabolite” that competitively inhibits α-KG dependent dioxygenases resulting in various widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Despite the growing understanding of IDH mutant gliomas, the development of effective therapies has proved challenging in part due to the scarcity of endogenous mutant in vivo models. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma model which rapidly grows in vivo, produces 2-HG and exhibits DNA hypermethylation. Using this model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumor regression was observed at 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results have implications for clinical trials of demethylating agents for patients with IDH mutated gliomas. PMID:24077805

  7. 5-azacytidine reduces methylation, promotes differentiation and induces tumor regression in a patient-derived IDH1 mutant glioma xenograft.

    Borodovsky, Alexandra; Salmasi, Vafi; Turcan, Sevin; Fabius, Armida W M; Baia, Gilson S; Eberhart, Charles G; Weingart, Jon D; Gallia, Gary L; Baylin, Stephen B; Chan, Timothy A; Riggins, Gregory J

    2013-10-01

    Somatic mutations in Isocitrate Dehydrogenase 1 (IDH1) are frequent in low grade and progressive gliomas and are characterized by the production of 2-hydroxyglutarate (2-HG) from α-ketoglutarate by the mutant enzyme. 2-HG is an "oncometabolite" that competitively inhibits α-KG dependent dioxygenases resulting in various widespread cellular changes including abnormal hypermethylation of genomic DNA and suppression of cellular differentiation. Despite the growing understanding of IDH mutant gliomas, the development of effective therapies has proved challenging in part due to the scarcity of endogenous mutant in vivo models. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma model which rapidly grows in vivo, produces 2-HG and exhibits DNA hypermethylation. Using this model, we have demonstrated the preclinical efficacy and mechanism of action of the FDA approved demethylating drug 5-azacytidine in vivo. Long term administration of 5-azacytidine resulted in reduction of DNA methylation of promoter loci, induction of glial differentiation, reduction of cell proliferation and a significant reduction in tumor growth. Tumor regression was observed at 14 weeks and subsequently showed no signs of re-growth at 7 weeks despite discontinuation of therapy. These results have implications for clinical trials of demethylating agents for patients with IDH mutated gliomas. PMID:24077805

  8. Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

    Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

  9. Lysine-specific histone demethylase 1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.

    Sun, Hao; Liang, Lining; Li, Yuan; Feng, Chengqian; Li, Lingyu; Zhang, Yixin; He, Songwei; Pei, Duanqing; Guo, Yunqian; Zheng, Hui

    2016-01-01

    Lysine-specific histone demethylase 1 (LSD1) regulates histone methylation and influences the epigenetic state of cells during the generation of induced pluripotent stem cells (iPSCs). Here we reported that LSD1 inhibition via shRNA or specific inhibitor, tranylcypromine, promoted reprogramming at early stage via two mechanisms. At early stage of reprogramming, LSD1 inhibition increased the retrovirus-mediated exogenous expression of Oct4, Klf4, and Sox2 by blocking related H3K4 demethylation. Since LSD1 inhibition still promoted reprogramming even when iPSCs were induced with small-molecule compounds in a virus-free system, additional mechanisms should be involved. When RNA-seq was used for analysis, it was found that LSD1 inhibition reversed some gene expression changes induced by OKS, which subsequently promoted reprogramming. For example, by partially rescuing the decreased expression of Hif1α, LSD1 inhibition reversed the up-regulation of genes in oxidative phosphorylation pathway and the down-regulation of genes in glycolysis pathway. Such effects facilitated the metabolic switch from oxidative phosphorylation to glycolysis and subsequently promoted iPSCs induction. In addition, LSD1 inhibition also promoted the conversion from pre-iPSCs to iPSCs by facilitating the similar metabolic switch. Therefore, LSD1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch. PMID:27481483

  10. Epigenetic silencing of the 3p22 tumor suppressor DLEC1 by promoter CpG methylation in non-Hodgkin and Hodgkin lymphomas

    Wang Zhaohui

    2012-10-01

    Full Text Available Abstract Background Inactivaion of tumor suppressor genes (TSGs by promoter CpG methylation frequently occurs in tumorigenesis, even in the early stages, contributing to the initiation and progression of human cancers. Deleted in lung and esophageal cancer 1 (DLEC1, located at the 3p22-21.3 TSG cluster, has been identified frequently silenced by promoter CpG methylation in multiple carcinomas, however, no study has been performed for lymphomas yet. Methods We examined the expression of DLEC1 by semi-quantitative reverse transcription (RT-PCR, and evaluated the promoter methylation of DLEC1 by methylation-specific PCR (MSP and bisulfite genomic sequencing (BGS in common lymphoma cell lines and tumors. Results Here we report that DLEC1 is readily expressed in normal lymphoid tissues including lymph nodes and PBMCs, but reduced or silenced in 70% (16/23 of non-Hodgkin and Hodgkin lymphoma cell lines, including 2/6 diffuse large B-cell (DLBCL, 1/2 peripheral T cell lymphomas, 5/5 Burkitt, 6/7 Hodgkin and 2/3 nasal killer (NK/T-cell lymphoma cell lines. Promoter CpG methylation was frequently detected in 80% (20/25 of lymphoma cell lines and correlated with DLEC1 downregulation/silencing. Pharmacologic demethylation reversed DLEC1 expression in lymphoma cell lines along with concomitant promoter demethylation. DLEC1 methylation was also frequently detected in 32 out of 58 (55% different types of lymphoma tissues, but not in normal lymph nodes. Furthermore, DLEC1 was specifically methylated in the sera of 3/13 (23% Hodgkin lymphoma patients. Conclusions Thus, methylation-mediated silencing of DLEC1 plays an important role in multiple lymphomagenesis, and may serve as a non-invasive tumor marker for lymphoma diagnosis.