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1
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Gassenmaier M, Rentschler M, Fehrenbacher B, Eigentler TK, Ikenberg K, Kosnopfel C, Sinnberg T, Niessner H, Bösmüller H, Wagner NB, Schaller M, Garbe C, Röcken M. Expression of DNA Methyltransferase 1 Is a Hallmark of Melanoma, Correlating with Proliferation and Response to B-Raf and Mitogen-Activated Protein Kinase Inhibition in Melanocytic Tumors. THE AMERICAN JOURNAL OF PATHOLOGY 2020; 190:2155-2164. [PMID: 32679231 DOI: 10.1016/j.ajpath.2020.07.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Revised: 06/29/2020] [Accepted: 07/03/2020] [Indexed: 12/11/2022]
Abstract
Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.
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Affiliation(s)
| | | | - Birgit Fehrenbacher
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Thomas K Eigentler
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Kristian Ikenberg
- Institute of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland
| | - Corinna Kosnopfel
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Tobias Sinnberg
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Heike Niessner
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Hans Bösmüller
- Institute of Pathology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Nikolaus B Wagner
- Department of Dermatology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland
| | - Martin Schaller
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Claus Garbe
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
| | - Martin Röcken
- Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany
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2
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Cui J, Liu N, Chang Z, Gao Y, Bao M, Xie Y, Xu W, Liu X, Jiang S, Liu Y, Shi R, Xie W, Jia X, Shi J, Ren C, Gong K, Zhang C, Bade R, Shao G, Ji X. Exosomal MicroRNA-126 from RIPC Serum Is Involved in Hypoxia Tolerance in SH-SY5Y Cells by Downregulating DNMT3B. MOLECULAR THERAPY-NUCLEIC ACIDS 2020; 20:649-660. [PMID: 32380415 PMCID: PMC7210387 DOI: 10.1016/j.omtn.2020.04.008] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 04/18/2020] [Accepted: 04/22/2020] [Indexed: 12/13/2022]
Abstract
Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.
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Affiliation(s)
- Junhe Cui
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Na Liu
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Zhehan Chang
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Yongsheng Gao
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Mulan Bao
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Yabin Xie
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Wenqiang Xu
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Xiaolei Liu
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Shuyuan Jiang
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - You Liu
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Rui Shi
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Wei Xie
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Xiaoe Jia
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC
| | - Jinghua Shi
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Changhong Ren
- Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC
| | - Kerui Gong
- Department of Oral and Maxillofacial Surgery, University of California San Francisco, San Francisco, CA, USA
| | - Chunyang Zhang
- Department of Neurosurgery, The First Affiliated Hospital of Baotou Medical College, Inner Mongolia, China
| | - Rengui Bade
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC.
| | - Guo Shao
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, PRC; Biomedicine Research Center, Basic Medical College and Baotou Medical College of the Neuroscience Institute, Baotou Medical College, Inner Mongolia, PRC; Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC; Department of Neurosurgery, The First Affiliated Hospital of Baotou Medical College, Inner Mongolia, China.
| | - Xunming Ji
- Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, PRC.
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Ye F, Huang J, Wang H, Luo C, Zhao K. Targeting epigenetic machinery: Emerging novel allosteric inhibitors. Pharmacol Ther 2019; 204:107406. [PMID: 31521697 DOI: 10.1016/j.pharmthera.2019.107406] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/04/2019] [Indexed: 12/13/2022]
Abstract
Epigenetics has emerged as an extremely exciting fast-growing area of biomedical research in post genome era. Epigenetic dysfunction is tightly related with various diseases such as cancer and aging related degeneration, potentiating epigenetics modulators as important therapeutics targets. Indeed, inhibitors of histone deacetylase and DNA methyltransferase have been approved for treating blood tumor malignancies, whereas inhibitors of histone methyltransferase and histone acetyl-lysine recognizer bromodomain are in clinical stage. However, it remains a great challenge to discover potent and selective inhibitors by targeting catalytic site, as the same subfamily of epigenetic enzymes often share high sequence identity and very conserved catalytic core pocket. It is well known that epigenetic modifications are usually carried out by multi-protein complexes, and activation of catalytic subunit is often tightly regulated by other interactive protein component, especially in disease conditions. Therefore, it is not unusual that epigenetic complex machinery may exhibit allosteric regulation site induced by protein-protein interactions. Targeting allosteric site emerges as a compelling alternative strategy to develop epigenetic drugs with enhanced druggability and pharmacological profiles. In this review, we highlight recent progress in the development of allosteric inhibitors for epigenetic complexes through targeting protein-protein interactions. We also summarized the status of clinical applications of those inhibitors. Finally, we provide perspectives of future novel allosteric epigenetic machinery modulators emerging from otherwise undruggable single protein target.
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Affiliation(s)
- Fei Ye
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, China; College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018; Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
| | - Jing Huang
- University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Hongbo Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, China; Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China.
| | - Cheng Luo
- University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Department of Pharmacy, Guizhou University of Traditional Chinese Medicine, South Dong Qing Road, Guizhou 550025, China.
| | - Kehao Zhao
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, China; Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China.
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4
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Chovanec M, Taza F, Kalra M, Hahn N, Nephew KP, Spinella MJ, Albany C. Incorporating DNA Methyltransferase Inhibitors (DNMTis) in the Treatment of Genitourinary Malignancies: A Systematic Review. Target Oncol 2019; 13:49-60. [PMID: 29230671 DOI: 10.1007/s11523-017-0546-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Inhibition of DNA methyltransferases (DNMTs) has emerged as a novel treatment strategy in solid tumors. Aberrant hypermethylation in promoters of critical tumor suppressor genes is the basis for the idea that treatment with hypomethylating agents may lead to the restoration of a "normal" epigenome and produce clinically meaningful therapeutic outcomes. The aim of this review article is to summarize the current state of knowledge of DNMT inhibitors in the treatment of genitourinary malignancies. The efficacy of these agents in genitourinary malignancies was reported in a number of studies and suggests a role of induced DNA hypomethylation in overcoming resistance to conventional cytotoxic treatments. The clinical significance of these findings should be further investigated.
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Affiliation(s)
- Michal Chovanec
- Division of Hematology/Oncology, Indiana University Simon Cancer Center, Indianapolis, IN, USA.
- 2nd Department of Oncology, Faculty of Medicine, Comenius University and National Cancer Institute, Bratislava, Slovakia.
| | - Fadi Taza
- Division of Hematology/Oncology, Indiana University Simon Cancer Center, Indianapolis, IN, USA
| | - Maitri Kalra
- Division of Hematology/Oncology, Indiana University Simon Cancer Center, Indianapolis, IN, USA
| | - Noah Hahn
- The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Kenneth P Nephew
- Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA
| | - Michael J Spinella
- Department of Comparative Biosciences, the University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Costantine Albany
- Division of Hematology/Oncology, Indiana University Simon Cancer Center, Indianapolis, IN, USA
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5
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Tian XL, Jiang SY, Zhang XL, Yang J, Cui JH, Liu XL, Gong KR, Yan SC, Zhang CY, Shao G. Potassium bisperoxo (1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases. Neural Regen Res 2019; 14:826-833. [PMID: 30688268 PMCID: PMC6375031 DOI: 10.4103/1673-5374.249230] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 09/25/2018] [Indexed: 01/08/2023] Open
Abstract
Bisperoxo (1,10-phenanthroline) oxovanadate (BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases (DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells (HT22) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT22 cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT22 cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT22 cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.
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Affiliation(s)
- Xiao-Li Tian
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
| | - Shu-Yuan Jiang
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Xiao-Lu Zhang
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
| | - Jie Yang
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Jun-He Cui
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Xiao-Lei Liu
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Ke-Rui Gong
- Department of Oral and Maxillofacial Surgery, University of California San Francsico, San Francisco, CA, USA
| | - Shao-Chun Yan
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Chun-Yang Zhang
- Department of Neurosurgery, the First Affiliated Hospital of Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
| | - Guo Shao
- Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
- Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
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Overexpression of DNMT3A promotes proliferation and inhibits differentiation of porcine intramuscular preadipocytes by methylating p21 and PPARg promoters. Gene 2019; 696:54-62. [DOI: 10.1016/j.gene.2019.02.029] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Revised: 01/21/2019] [Accepted: 02/01/2019] [Indexed: 12/12/2022]
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7
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Moghaddaskho F, Eyvani H, Ghadami M, Tavakkoly-Bazzaz J, Alimoghaddam K, Ghavamzadeh A, Ghaffari SH. Demethylation and alterations in the expression level of the cell cycle-related genes as possible mechanisms in arsenic trioxide-induced cell cycle arrest in human breast cancer cells. Tumour Biol 2017; 39:1010428317692255. [PMID: 28218039 DOI: 10.1177/1010428317692255] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Arsenic trioxide (As2O3) has been used clinically as an anti-tumor agent. Its mechanisms are mostly considered to be the induction of apoptosis and cell cycle arrest. However, the detailed molecular mechanisms of its anti-cancer action through cell cycle arrest are poorly known. Furthermore, As2O3 has been shown to be a potential DNA methylation inhibitor, inducing DNA hypomethylation. We hypothesize that As2O3 may affect the expression of cell cycle regulatory genes by interfering with DNA methylation patterns. To explore this, we examined promoter methylation status of 24 cell cycle genes in breast cancer cell lines and in a normal breast tissue sample by methylation-specific polymerase chain reaction and/or restriction enzyme-based methods. Gene expression level and cell cycle distribution were quantified by real-time polymerase chain reaction and flow cytometric analyses, respectively. Our methylation analysis indicates that only promoters of RBL1 (p107), RASSF1A, and cyclin D2 were aberrantly methylated in studied breast cancer cell lines. As2O3 induced CpG island demethylation in promoter regions of these genes and restores their expression correlated with DNA methyltransferase inhibition. As2O3 also induced alterations in messenger RNA expression of several cell cycle-related genes independent of demethylation. Flow cytometric analysis revealed that the cell cycle arrest induced by As2O3 varied depending on cell lines, MCF-7 at G1 phase and both MDA-MB-231 and MDA-MB-468 cells at G2/M phase. These changes at transcriptional level of the cell cycle genes by the molecular mechanisms dependent and independent of demethylation are likely to represent the mechanisms of cell cycle redistribution in breast cancer cells, in response to As2O3 treatment.
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Affiliation(s)
- Farima Moghaddaskho
- 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.,2 Medical Genetics Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Haniyeh Eyvani
- 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.,2 Medical Genetics Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mohsen Ghadami
- 2 Medical Genetics Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Javad Tavakkoly-Bazzaz
- 2 Medical Genetics Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Kamran Alimoghaddam
- 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Ardeshir Ghavamzadeh
- 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Seyed H Ghaffari
- 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
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8
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Xi Q, Gao N, Yang Y, Ye W, Zhang B, Wu J, Jiang G, Zhang X. Anticancer drugs induce hypomethylation of the acetylcholinesterase promoter via a phosphorylated-p38-DNMT1-AChE pathway in apoptotic hepatocellular carcinoma cells. Int J Biochem Cell Biol 2015; 68:21-32. [DOI: 10.1016/j.biocel.2015.08.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 08/17/2015] [Accepted: 08/17/2015] [Indexed: 10/23/2022]
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9
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How J, Minden MD, Brian L, Chen EX, Brandwein J, Schuh AC, Schimmer AD, Gupta V, Webster S, Degelder T, Haines P, Stayner LA, McGill S, Wang L, Piekarz R, Wong T, Siu LL, Espinoza-Delgado I, Holleran JL, Egorin MJ, Yee KWL. A phase I trial of two sequence-specific schedules of decitabine and vorinostat in patients with acute myeloid leukemia. Leuk Lymphoma 2015; 56:2793-802. [PMID: 25682963 PMCID: PMC4688006 DOI: 10.3109/10428194.2015.1018248] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
This phase I trial evaluated two schedules of escalating vorinostat in combination with decitabine every 28 days: (i) sequential or (ii) concurrent. There were three dose-limiting toxicities: grade 3 fatigue and generalized muscle weakness on the sequential schedule (n = 1) and grade 3 fatigue on the concurrent schedule (n = 2). The maximum tolerated dose was not reached on both planned schedules. The overall response rate (ORR) was 23% (three complete response [CR], two CR with incomplete incomplete blood count recovery [CRi], one partial response [PR] and two morphological leukemic free state [MLFS]). The ORR for all and previously untreated patients in the sequential arm was 13% (one CRi; one MLFS) and 0% compared to 30% (three CR; one CRi; one PR; one MLFS) and 36% in the concurrent arm (p = 0.26 for both), respectively. Decitabine plus vorinostat was safe and has clinical activity in patients with previously untreated acute myeloid leukemia. Responses appear higher with the concurrent dose schedule. Cumulative toxicities may limit long-term usage on the current dose/schedules.
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Affiliation(s)
- Jonathan How
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Mark D Minden
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Leber Brian
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Eric X Chen
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | | | - Andre C Schuh
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | | | - Vikas Gupta
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Sheila Webster
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Tammy Degelder
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Patricia Haines
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | | | - Shauna McGill
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Lisa Wang
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Richard Piekarz
- b Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute , Bethesda , MD , USA
| | - Tracy Wong
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Lillian L Siu
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
| | - Igor Espinoza-Delgado
- b Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute , Bethesda , MD , USA
| | - Julianne L Holleran
- c Departments of Medicine and Pharmacology and Cancer Institute , University of Pittsburgh , Pittsburgh , PA , USA
| | - Merrill J Egorin
- c Departments of Medicine and Pharmacology and Cancer Institute , University of Pittsburgh , Pittsburgh , PA , USA
| | - Karen W L Yee
- a Princess Margaret Phase I Consortium , Toronto , ON , Canada
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10
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Williams KE, Anderton DL, Lee MP, Pentecost BT, Arcaro KF. High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines. Epigenetics 2013; 9:297-307. [PMID: 24225485 DOI: 10.4161/epi.27111] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2-11 and TMX2-28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2-11 had 4000 hypermethylated sites and ERα-negative TMX2-28 had over 33 000. Analysis of CpG sites altered in both TMX2-11 and TMX2-28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2ꞌdeoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2-28. A similar relationship between methylation and expression was not detected in TMX2-11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.
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Affiliation(s)
- Kristin E Williams
- Molecular & Cellular Biology Graduate Program; Department of Veterinary & Animal Sciences; University of Massachusetts; Amherst, MA USA
| | | | - Maxwell P Lee
- Center for Cancer Research; National Institutes of Health; Bethesda, MD USA
| | | | - Kathleen F Arcaro
- Molecular & Cellular Biology Graduate Program; Department of Veterinary & Animal Sciences; University of Massachusetts; Amherst, MA USA
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11
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ATM mediates pRB function to control DNMT1 protein stability and DNA methylation. Mol Cell Biol 2013; 33:3113-24. [PMID: 23754744 DOI: 10.1128/mcb.01597-12] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.
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12
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Yuan B, Zhang J, Wang H, Xiong L, Cai Q, Wang T, Jacobsen S, Pradhan S, Wang Y. 6-Thioguanine reactivates epigenetically silenced genes in acute lymphoblastic leukemia cells by facilitating proteasome-mediated degradation of DNMT1. Cancer Res 2011; 71:1904-11. [PMID: 21239472 DOI: 10.1158/0008-5472.can-10-3430] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Thiopurines including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine are effective anticancer agents with remarkable success in clinical practice, especially in effective treatment of acute lymphoblastic leukemia (ALL). (S)G is understood to act as a DNA hypomethylating agent in ALL cells, however, the underlying mechanism leading to global cytosine demethylation remains unclear. Here we report that (S)G treatment results in reactivation of epigenetically silenced genes in T leukemia cells. Bisulfite genomic sequencing revealed that (S)G treatment universally elicited demethylation in the promoters and/or first exons of the genes that were reactivated. (S)G treatment also attenuated the expression of histone lysine-specific demethylase 1 (LSD1), thereby stimulating lysine methylation of the DNA methylase DNMT1 and triggering its degradation via the ubiquitin-proteasomal pathway. Taken together, our findings reveal a previously uncharacterized but vital mechanistic link between (S)G treatment and DNA hypomethylation.
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Affiliation(s)
- Bifeng Yuan
- Department of Chemistry, University of California, Riverside, California 92521-0403, USA
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13
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Evdokimov AA, Zinoviev VV, Kuznetsov VV, Netesova NA, Malygin EG. Design of oligonucleotide inhibitors for human DNA methyltransferase 1. Mol Biol 2009. [DOI: 10.1134/s0026893309030108] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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14
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Saavedra OM, Isakovic L, Llewellyn DB, Zhan L, Bernstein N, Claridge S, Raeppel F, Vaisburg A, Elowe N, Petschner AJ, Rahil J, Beaulieu N, MacLeod AR, Delorme D, Besterman JM, Wahhab A. SAR around (l)-S-adenosyl-l-homocysteine, an inhibitor of human DNA methyltransferase (DNMT) enzymes. Bioorg Med Chem Lett 2009; 19:2747-51. [DOI: 10.1016/j.bmcl.2009.03.113] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2009] [Revised: 03/23/2009] [Accepted: 03/25/2009] [Indexed: 10/21/2022]
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15
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Plummer R, Vidal L, Griffin M, Lesley M, de Bono J, Coulthard S, Sludden J, Siu LL, Chen EX, Oza AM, Reid GK, McLeod AR, Besterman JM, Lee C, Judson I, Calvert H, Boddy AV. Phase I study of MG98, an oligonucleotide antisense inhibitor of human DNA methyltransferase 1, given as a 7-day infusion in patients with advanced solid tumors. Clin Cancer Res 2009; 15:3177-83. [PMID: 19383817 DOI: 10.1158/1078-0432.ccr-08-2859] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE To assess the safety and tolerability, pharmacokinetics, and early evidence of antitumor activity of escalating doses of MG98, an antisense oligonucleotide to DNA methyltransferase 1 (DNMT1), which has been shown to reduce CpG island methylation and allow reexpression of tumor suppressor genes in vitro. EXPERIMENTAL DESIGN In this phase I, open-label study, patients with advanced solid malignancies were treated with escalating doses of MG98 administered as a continuous i.v. infusion over 7 days repeated every 14 days. Cohorts of three patients, which could be expanded to six patients, were studied. The maximum tolerated dose was defined as the highest dose at which no more than 33% of subjects experienced dose-limiting toxicity. Pharmacokinetic and pharmacodynamic parameters of MG98 were also characterized. RESULTS Thirty-three patients were treated at doses of 100 to 250 mg/m(2)/d MG98. MG98 was well tolerated with mild fatigue and myalgia, dose-limiting toxicity was asymptomatic transaminitis, and the maximum tolerated dose was 200 mg/m(2)/d. One patient achieved a partial response and another prolonged disease stabilization. Plasma half-life of MG98 was short (2 hours), drug concentrations reaching a dose-dependent steady state during infusion with a volume of distribution equivalent to plasma volume. Suppression of DNMT1 expression was observed in 26 of 32 patients studied. CONCLUSIONS MG98 was well tolerated with early evidence of clinical activity. Proof of mechanism was observed and measurement of DNMT1 expression in peripheral blood mononuclear cells may be useful in future phase II development.
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Affiliation(s)
- Ruth Plummer
- Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK.
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16
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Tan HH, Porter AG. p21(WAF1) negatively regulates DNMT1 expression in mammalian cells. Biochem Biophys Res Commun 2009; 382:171-6. [PMID: 19275888 DOI: 10.1016/j.bbrc.2009.03.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2009] [Accepted: 03/01/2009] [Indexed: 10/21/2022]
Abstract
The expression of DNMT1, the major maintenance DNA methyltransferases, is critical in coordinating DNMT1 activity with biological processes and therefore must be tightly regulated in the cell cycle. Here, we report p21(WAF1) as a novel upstream regulator of DNMT1 expression. Ectopic expression of p21(WAF1) or TSA-mediated p21(WAF1) induction inhibits DNMT1 at the transcriptional level, and this observation consistently coincides with a reduction in p300. Furthermore, siRNA-mediated p300 knockdown significantly abolishes DNMT1 mRNA levels, demonstrating the dependence of DNMT1 expression on p300. Consistent with this, p300 enhances transactivation of DNMT1 promoter 340bp upstream of the initiation start site harboring the E2F1 and Sp1/3 binding sites. Collectively, we identified p300 as a crucial transcription regulator for DNMT1. We proposed that the reduction in p300 following p21(WAF1) up-regulation contributes to DNMT1 down-regulation. This novel p21(WAF1)-p300-DNMT1 pathway may play a pivotal role to ensure regulated DNMT1 expression and DNA methylation in mammalian cell division.
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Affiliation(s)
- Hwee Hong Tan
- Cell Death and Human Disease Group, Division of Cancer and Developmental Cell Biology, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, Singapore 138673, Singapore.
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17
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Szyf M. DNA demethylation and cancer metastasis: therapeutic implications. Expert Opin Drug Discov 2008; 3:519-31. [DOI: 10.1517/17460441.3.5.519] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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18
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Histone deacetylase inhibitor depsipeptide activates silenced genes through decreasing both CpG and H3K9 methylation on the promoter. Mol Cell Biol 2008; 28:3219-35. [PMID: 18332107 DOI: 10.1128/mcb.01516-07] [Citation(s) in RCA: 103] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di- and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1alpha and HP1beta) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter.
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19
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DNA methyltransferase 1 knockdown induces silenced CDH1 gene reexpression by demethylation of methylated CpG in hepatocellular carcinoma cell line SMMC-7721. Eur J Gastroenterol Hepatol 2007; 19:952-61. [PMID: 18049164 DOI: 10.1097/meg.0b013e3282c3a89e] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality in the world; however, the molecular mechanisms leading to hepatocyte transformation, especially in epigenetic mechanisms (such as DNA methylation) are still poorly understood. DNA methyltransferase 1 (DNMT1) is the predominant maintenance methyltransferase gene required to maintain DNA methylation patterns in mammalian cells. AIM AND METHODS To explore the role of DNMT1 in the regulation of expression of tumor-related genes in human HCC cells via DNA methylation of the regulatory CpG islands, we stably transfected expression constructs containing small interfering RNA (siRNA) of DNMT1 into the human HCC cell line, SMMC-7721. RESULTS RNA interference knocked down specific DNMT1 protein expression, resulting in the demethylated promoter of CDH1 and the reexpression of CDH1 in 7721-pMT1. By contrast, promoter methylation and lack of gene expression were maintained when the cell lines had control constructs. Knock down of DNMT1 expression by siRNA induced the promoter of CDH1 demethylation and upregulated CDH1 transcription. High-density oligonucleotide gene expression microarrays were used to examine the effects of DNMT1 knock down on human HCC cells (SMMC-7721); these showed that a number of genes were induced in the DNMT1 knock down cell lines, including some important tumor-related genes such as PDCD4, DCN and PTGES except CDH1. Only approximately 78% of the induced genes have CpG islands within their 5' regions, suggesting that certain genes activated by DNMT1 siRNA might not have resulted from the direct inhibition of promoter methylation. CONCLUSION In hepatocellular carcinoma, DNMT1 is necessary to maintain the methylation of CpG islands in certain tumor-related genes.
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20
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The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Oncogene 2007; 27:2187-97. [PMID: 17934516 DOI: 10.1038/sj.onc.1210855] [Citation(s) in RCA: 136] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.
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21
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Tamm I, Wagner M, Schmelz K. Decitabine activates specific caspases downstream of p73 in myeloid leukemia. Ann Hematol 2007; 84 Suppl 1:47-53. [PMID: 16193303 DOI: 10.1007/s00277-005-0013-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The demethylating effect of 5-aza-2' deoxycytidine (decitabine, DAC) has been well characterized. The molecular events downstream of methylation inhibition are less well known. Here, DAC was shown to induce apoptosis in acute myeloid leukemia (AML) cells (p53 mutant and wild type) but not in epithelial or normal peripheral blood mononuclear cells. Apoptosis was characterized by activation of the mitochondrial but not the receptor death pathway, as demonstrated by the release of cytochrome c and loss of mitochondrial membrane potential. Western blotting and enzyme assays showed that caspase-3, but not caspase-6 or caspase-8, were activated. Decitabine induced expression of the cell cycle inhibitor p21, arresting AML cell lines in G1 of the cell cycle. Expression of p21 was induced irrespective of the methylation status of its promoter, mediated instead via reexpression of the tumor suppressor p73, an upstream regulator of p21. The promoter of p73 was hypermethylated in AML cell lines in vitro and in primary AML cells ex vivo but not in DAC-resistant epithelial cells. In conclusion, DAC acts on leukemic myeloid cells via caspase activation, which may be dependent on demethylation of the hypermethylated p73 promoter and consequent reexpression of p73.
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Affiliation(s)
- Ingo Tamm
- Department of Hematology and Oncology, Universitätsmedizin Berlin Charité, Campus Virchow, Berlin, Germany.
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22
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You JS, Kang JK, Lee EK, Lee JC, Lee SH, Jeon YJ, Koh DH, Ahn SH, Seo DW, Lee HY, Cho EJ, Han JW. Histone deacetylase inhibitor apicidin downregulates DNA methyltransferase 1 expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells. Oncogene 2007; 27:1376-86. [PMID: 17828306 DOI: 10.1038/sj.onc.1210776] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.
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Affiliation(s)
- J S You
- Department of Biochemistry and Molecular Biology, College of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea
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Abstract
The transcriptional regulation of p16INK4a is essential for cellular aging and oncogenic stress response. This regulation involves p16INK4a transcriptional activators such as proteins Ets1 and 2 or E47. The binding of these proteins to INK4a promoter can be inhibited by proteins Id-1 or -4 after heterodimer formation. The transcriptional inhibition of p16INK4a includes also the transcriptional repression by Bmi-1, and an epigenetic regulation which appears complex and remains incompletely understood. Actually, INK4a promoter and exon1 present a CpG island which can be methylated on cytosines by DNA methyltransferases. This DNA methylation is preceded by the lysine 9 histone H3 methylation and by the deacetylation of histone H4 both involved in gene silencing. Indeed, RNA Helicase A might protect INK4a against methylation of CpG island. Furthermore, chromatin remodelling involving SWI/SNF complex, antagonist to Bmi-1, might activate INK4a expression. The analysis of INK4a regulation mechanisms and the comprehension of the epigenetic modulation of its expression may allow us to develop a rational use of new anti-neoplastic agents.
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Affiliation(s)
- Wei Wen Chien
- Laboratoire de cytologie analytique, Faculte de medecine, France
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24
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Monier K, Mouradian S, Sullivan KF. DNA methylation promotes Aurora-B-driven phosphorylation of histone H3 in chromosomal subdomains. J Cell Sci 2007; 120:101-14. [PMID: 17164288 DOI: 10.1242/jcs.03326] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Confinement of enzymatic reactions to nuclear and chromosomal subdomains regulates functional organization of the nucleus. Aurora-B kinase regulates cell-cycle-dependent phosphorylation of chromosomal substrates through sequential localization to a series of sites on chromosomes and the mitotic spindle. In G2 nuclei, Aurora-B recruitment to heterochromatin restricts histone H3S10 phosphorylation to a domain around centromeres (pericentromeres). However, no intrinsic chromosomal determinants have been implicated in Aurora-B recruitment to interphase pericentromeres. Using cyclin B1 as a cell-cycle marker, we found that the great majority of nuclei exhibiting H3S10 phosphorylated foci were positive for cyclin B1, thus revealing that H3S10 phosphorylation arises at pericentromeres during late S phase and persists in G2. By immunofluorescent in situ hybridization, Aurora-B and H3S10 phosphorylated foci were found more frequently at larger pericentromeres than at smaller ones, revealing a preferential phosphorylation of pericentromeres, exhibiting a high density of methyl cytosines. Disruption of DNA methylation inhibited pericentromeric Aurora-B targeting and H3S10 phosphorylation in G2 nuclei, thus demonstrating the role of DNA methylation in Aurora-B targeting to pericentromeres. These results favour the idea that DNA methylation maintains a local environment essential for regulating the functional properties of sub-chromosomal domains during S-G2 progression.
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Affiliation(s)
- Karine Monier
- The Scripps Research Institute, Department of Cell Biology, CB163, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
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25
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Olave IA, Doneanu C, Fang X, Stamatoyannopoulos G, Li Q. Purification and identification of proteins that bind to the hereditary persistence of fetal hemoglobin -198 mutation in the gamma-globin gene promoter. J Biol Chem 2006; 282:853-62. [PMID: 17114178 PMCID: PMC2819221 DOI: 10.1074/jbc.m610404200] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.
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MESH Headings
- Adult
- Animals
- Antibody Specificity
- Blotting, Western
- Cell Fractionation
- Cell Line, Tumor
- Chromatography, Affinity
- DNA (Cytosine-5-)-Methyltransferase 1
- DNA (Cytosine-5-)-Methyltransferases/immunology
- DNA (Cytosine-5-)-Methyltransferases/isolation & purification
- DNA (Cytosine-5-)-Methyltransferases/metabolism
- Fetal Hemoglobin/genetics
- Gene Expression Regulation, Developmental
- Globins/genetics
- Humans
- Leukemia, Erythroblastic, Acute
- Mass Spectrometry
- Mice
- Mice, Transgenic
- Nuclear Matrix-Associated Proteins/immunology
- Nuclear Matrix-Associated Proteins/isolation & purification
- Nuclear Matrix-Associated Proteins/metabolism
- Point Mutation
- Promoter Regions, Genetic/physiology
- Sp1 Transcription Factor/immunology
- Sp1 Transcription Factor/isolation & purification
- Sp1 Transcription Factor/metabolism
- Transcription Factors/immunology
- Transcription Factors/isolation & purification
- Transcription Factors/metabolism
- Transcription Factors, TFII/immunology
- Transcription Factors, TFII/isolation & purification
- Transcription Factors, TFII/metabolism
- Transcriptional Activation
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Affiliation(s)
- Ivan A. Olave
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington 98195
| | - Catalin Doneanu
- Mass Spectrometry Center, Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195
| | - Xiangdong Fang
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington 98195
| | - George Stamatoyannopoulos
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington 98195
| | - Qiliang Li
- Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington 98195
- To whom correspondence should be addressed: Div. of Medical Genetics, University of Washington, P. O. Box 357720, Seattle, WA 98195. Tel.: 206-616-4526; Fax: 206-616-4527;
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26
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Unterberger A, Andrews SD, Weaver ICG, Szyf M. DNA methyltransferase 1 knockdown activates a replication stress checkpoint. Mol Cell Biol 2006; 26:7575-86. [PMID: 17015478 PMCID: PMC1636877 DOI: 10.1128/mcb.01887-05] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.
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Affiliation(s)
- Alexander Unterberger
- Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec, Canada H3G 1Y6
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27
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Segura-Pacheco B, Perez-Cardenas E, Taja-Chayeb L, Chavez-Blanco A, Revilla-Vazquez A, Benitez-Bribiesca L, Duenas-González A. Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine. J Transl Med 2006; 4:32. [PMID: 16893460 PMCID: PMC1563479 DOI: 10.1186/1479-5876-4-32] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2006] [Accepted: 08/07/2006] [Indexed: 11/10/2022] Open
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 hypermethylation. 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 hypermethylation, 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.
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Affiliation(s)
- Blanca Segura-Pacheco
- Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonóma de Mexico, Instituto Nacional de Cancerología, Mexico
| | - Enrique Perez-Cardenas
- Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonóma de Mexico, Instituto Nacional de Cancerología, Mexico
| | - Lucia Taja-Chayeb
- Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonóma de Mexico, Instituto Nacional de Cancerología, Mexico
| | - Alma Chavez-Blanco
- Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonóma de Mexico, Instituto Nacional de Cancerología, Mexico
| | - Alma Revilla-Vazquez
- Lab. de Desarrollo de Metodos Analiticos, FES-Cuautitlan, UNAM, Cuautitlan Izcalli, Estado de Mexico, Mexico
| | - Luis Benitez-Bribiesca
- Unidad de Investigacion Medica en Enfermedades Oncologicas, Hospital de Oncologia, CMN-SXXI, IMSS, DF, Mexico
| | - Alfonso Duenas-González
- Unidad de Investigación Biomédica en Cáncer, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonóma de Mexico, Instituto Nacional de Cancerología, Mexico
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28
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Fang JY, Lu R, Mikovits JA, Cheng ZH, Zhu HY, Chen YX. Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1. Cancer Lett 2006; 233:124-30. [PMID: 16473673 DOI: 10.1016/j.canlet.2005.03.005] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2004] [Revised: 03/04/2005] [Accepted: 03/06/2005] [Indexed: 11/28/2022]
Abstract
Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methyltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (hMLH1) and human Mut S homologue 2 (hMSH2), was monitored by real-time RT-PCR. The methylation status of hMLH1 and hMSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1, hMSH2 and hMLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of hMLH1 and hMSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of hMSH2 (P<0.05) and hMLH1 (P=0.064) in SW1116 cells.
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Affiliation(s)
- Jing Yuan Fang
- Shanghai Second Medical University Renji Hospital, Shanghai Institute of Digestive Disease, 145 Shandong Zhong Road, Shanghai 200001, China.
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29
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Loriot A, De Plaen E, Boon T, De Smet C. Transient down-regulation of DNMT1 methyltransferase leads to activation and stable hypomethylation of MAGE-A1 in melanoma cells. J Biol Chem 2006; 281:10118-26. [PMID: 16497664 DOI: 10.1074/jbc.m510469200] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression.
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Affiliation(s)
- Axelle Loriot
- Ludwig Institute for Cancer Research, Brussels and Cellular Genetics Unit, Université Catholique de Louvain, 74 Avenue Hippocrate, B1200 Brussels, Belgium
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30
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Alvarez-Nuñez F, Bussaglia E, Mauricio D, Ybarra J, Vilar M, Lerma E, de Leiva A, Matias-Guiu X. PTEN promoter methylation in sporadic thyroid carcinomas. Thyroid 2006; 16:17-23. [PMID: 16487009 DOI: 10.1089/thy.2006.16.17] [Citation(s) in RCA: 120] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The tumor-suppressor gene PTEN/MMAC1, on chromosome 10q23.3, has been implicated in an important number of human tumors, such as thyroid carcinomas. PTEN somatic mutations occur in sporadic tumors of the endometrium, brain, prostate, or melanomas, while germline mutations predispose to development of the multiple hamartoma syndromes (i.e., Cowden's disease and Bannayan-Zonana syndrome). Activation of the two alleles of PTEN is required for its tumor-suppression role. Because the frequency of PTEN suppression in thyroid tumors exceeds that of PTEN mutations or deletions, it is very likely that epigenetic mechanisms, such as promoter hypermethylation, may account for its inactivation in a subset of tumors. The main aim of this study was to assess the frequency of promoter hypermethylation of PTEN in thyroid tumors. We studied frozen tissue samples from 46 papillary carcinomas, 7 follicular carcinomas, 6 follicular adenomas as well as 39 normal thyroid tissue samples. Methylation-specific polymerase-chain reaction (PCR) with three different sets of primers was used. Two of the primer sets were designed to avoid any interference with PTEN pseudogene promoter. PTEN promoter hypermethylation was detected in 21 of 46 (45.7%) papillary carcinomas, 6 of 7 follicular carcinomas, and 5 of 6 follicular adenomas. It was negative in all normal tissues. Negative immunohistochemical staining for PTEN was significantly associated with the presence of promoter hypermethylation (p < 0.001). These results show a high frequency of PTEN promoter hypermethylation, especially in follicular tumors, suggesting its possible role in thyroid tumorigenesis.
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Affiliation(s)
- Francisco Alvarez-Nuñez
- Laboratory of Experimental Endocrinology, Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
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31
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Roberti A, La Sala D, Cinti C. Multiple genetic and epigenetic interacting mechanisms contribute to clonally selection of drug-resistant tumors: Current views and new therapeutic prospective. J Cell Physiol 2006; 207:571-81. [PMID: 16250021 DOI: 10.1002/jcp.20515] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Successful treatment of cancer requires a clear understanding of drug-resistance mechanism. Cancer patient are often treated with standard protocols without considering individual difference in chemosensitivity, whereas the efficacy of anticancer drug varies widely among individual patients. Since chemosensitivity involves multiple interacting factors, it is not sufficient to investigate a single gene or factor to fix chemoresistance. Along with affecting disease progression, the synergism between genetic and epigenetic abnormalities can contribute to convert a sensible tumor cell in a resistant one. Unlike genetic changes, epigenetic changes are potentially reversible. Therefore, treatment with DNA methylation inhibitors can reactivate the expression of genes improperly methylated and can reverse many aspect of cancer phenotype such as drug resistance. The demethylating agents are used in the treatment of several kind of tumor, but toxicity and the potential outcome on the normal methylation patterns have always been concern of researchers and clinicals. It is necessary to create individual chemosensitivity-chemoresistance maps in order to identify the combination of drugs for optimized treatments. An overview on genetic and epigenetic events contributing to clonally selection of chemotherapeutic-resistant tumors is discussed.
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Affiliation(s)
- Annalisa Roberti
- Institute of Clinical Physiology (IFC), National Research Council (CNR), Siena, Italy
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32
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Novakovic P, Stempak JM, Sohn KJ, Kim YI. Effects of folate deficiency on gene expression in the apoptosis and cancer pathways in colon cancer cells. Carcinogenesis 2005; 27:916-24. [PMID: 16361273 DOI: 10.1093/carcin/bgi312] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Folate is a B vitamin, deficiency of which appears to increase the risk of developing several malignancies including colorectal cancer. In contrast to the cancer-promoting effect of folate deficiency in normal tissues, several lines of evidence indicate that folate depletion suppresses the progression of existing neoplasms and enhance the sensitivity of cancer cells to chemotherapy. Folate mediates the transfer of one-carbon necessary for the de novo biosynthesis of purines and thymidylate, and hence is an essential factor for DNA synthesis and repair, and the maintenance of DNA integrity and stability. Folate deficiency induces DNA strand breaks, increases uracil misincorporation into DNA, impairs DNA repair and appears to induce apoptosis. Although the effects of folate depletion on DNA integrity and apoptosis and on subsequent cancer development, progression and treatment in colonic epithelial cells have been well characterized, it is largely unknown at present how folate depletion modulates specific upstream genes in apoptosis and cancer pathways that regulate these processes. We therefore investigated the effects of folate depletion on expression of genes involved in apoptosis and cancer pathways in four human colon adenocarcinoma cell lines in an in vitro model of folate deficiency. Apoptosis and cancer pathway-specific mini-microarray were used to screen for differentially expressed genes in response to folate deficiency, and the expression of seven most notably and consistently affected genes was confirmed by real time RT-PCR. Our data suggest that folate deficiency affects the expression of key genes that are related to cell cycle control, DNA repair, apoptosis and angiogenesis in a cell-specific manner. Cell-specificity in gene expression changes in response to folate deficiency is likely due to significant differences in molecular and phenotypic characteristics, growth rates and intracellular folate concentrations among the four cell lines.
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Affiliation(s)
- Petar Novakovic
- Institute of Medical Science, Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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33
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Szyf M. DNA methylation and demethylation as targets for anticancer therapy. BIOCHEMISTRY (MOSCOW) 2005; 70:533-49. [PMID: 15948707 DOI: 10.1007/s10541-005-0147-7] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Cancer growth and metastasis require the coordinate change in gene expression of different sets of genes. While genetic alterations can account for some of these changes, it is becoming evident that many of the changes in gene expression observed are caused by epigenetic modifications. The epigenome consists of the chromatin and its modifications, the "histone code" as well as the pattern of distribution of covalent modifications of cytosines residing in the dinucleotide sequence CG by methylation. Although hypermethylation of tumor suppressor genes has attracted a significant amount of attention and inhibitors of DNA methylation were shown to activate methylated tumor suppressor genes and inhibit tumor growth, demethylation of critical genes plays a critical role in cancer as well. This review discusses the emerging role of demethylation in activation of pro-metastatic genes and the potential therapeutic implications of the demethylation machinery in metastasis.
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Affiliation(s)
- M Szyf
- Department of Pharmacology and Therapeutics, McGill University, Montreal PQ H3G 1Y6, Canada.
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34
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Lin TS, Lee H, Chen RA, Ho ML, Lin CY, Chen YH, Tsai YY, Chou MC, Cheng YW. An association of DNMT3b protein expression with P16INK4a promoter hypermethylation in non-smoking female lung cancer with human papillomavirus infection. Cancer Lett 2005; 226:77-84. [PMID: 16004934 DOI: 10.1016/j.canlet.2004.12.031] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2004] [Revised: 12/08/2004] [Accepted: 12/13/2004] [Indexed: 12/31/2022]
Abstract
Our recent report indicated that HPV infection may be associated with an increased frequency of p16INK4a promoter hypermethylation to cause p16 inactivation. In this study, we further speculated that the HPV infection may be linked with the expression of DNA methyltransferase (DNMT) protein in lung cancer patients and it was observed that an association of p16INK4a promoter hypermethylation with HPV infection existed, but only in female cases (P<0.0001). Interestingly, DNMT3b protein expression was significantly correlated with p16INK4a promoter hypermethylation (P=0.023) and HPV 16/18 infections (P<0.001), respectively. Moreover, the correlation between p16INK4a promoter hypermethylation and DNMT3b protein expression was exclusively seen in female cases (P=0.035). These results strongly suggested that the involvement of HPV infection in nonsmoking female lung tumorigenesis may be mediated, at least to a certain extent, through the increase of DNMT3b protein expression to cause p16INK4a promoter hypermethylation.
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Affiliation(s)
- Torng-Sen Lin
- Institute of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd, Taichung, Taiwan, ROC
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35
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Schmelz K, Wagner M, Dörken B, Tamm I. 5-Aza-2'-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia. Int J Cancer 2005; 114:683-95. [PMID: 15609309 DOI: 10.1002/ijc.20797] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (DeltaPsim). Activation of caspase-3 (but not -6 and -8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.
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MESH Headings
- Apoptosis
- Azacitidine/analogs & derivatives
- Azacitidine/pharmacology
- Blotting, Western
- Caspase 3
- Caspases/metabolism
- Cell Cycle
- Cell Cycle Proteins/biosynthesis
- Cell Line, Tumor
- CpG Islands
- Cyclin-Dependent Kinase Inhibitor p21
- Cytochromes c/metabolism
- DNA Methylation
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- Decitabine
- Epithelial Cells/cytology
- Epithelial Cells/metabolism
- Flow Cytometry
- G1 Phase
- Genes, Tumor Suppressor
- HL-60 Cells
- HeLa Cells
- Humans
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy
- Leukemia, Myeloid, Acute/drug therapy
- Leukocytes, Mononuclear/metabolism
- Membrane Potentials
- Microscopy, Fluorescence
- Mitochondria/metabolism
- Myelodysplastic Syndromes/drug therapy
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Promoter Regions, Genetic
- RNA/chemistry
- RNA, Messenger/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Sulfites/chemistry
- Time Factors
- Transfection
- Tumor Protein p73
- Tumor Suppressor Protein p53/metabolism
- Tumor Suppressor Proteins
- U937 Cells
- Up-Regulation
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Affiliation(s)
- Karin Schmelz
- Department of Hematology and Oncology, Universitätsmedizin Berlin, Charité, Campus Virchow, Berlin, Germany
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36
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Schmelz K, Sattler N, Wagner M, Lübbert M, Dörken B, Tamm I. Induction of gene expression by 5-Aza-2'-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms. Leukemia 2005; 19:103-11. [PMID: 15510208 DOI: 10.1038/sj.leu.2403552] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR, decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after 5-Aza-CdR treatment in AML. In the AML cell line OCI-AML2, Aza-CdR induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (> or =2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with 5-Aza-CdR ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with 5-Aza-CdR. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5' region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected 5-Aza-CdR-inducible genes. We detected hypermethylation exclusively in the 5' region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P< or =0.004). In contrast, all other analyzed 5-Aza-CdR-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of 5-Aza-CdR.
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Affiliation(s)
- K Schmelz
- Charité, Department of Hematology and Oncology, Virchow-Clinic, Internal Medicine, Universitätsmedizin Berlin, Campus Virchow, Augustenburger Platz 1, 13353 Berlin, Germany
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Abstract
Cancer growth and metastasis requires reprogramming of the expression of multiple genes. The epigenome, which is comprised of chromatin and the patterns of DNA methylation, sets up and maintains gene expression programs. As expected from the broad changes in gene expression in cancer, which are characterized by both silencing and activation of multiple genes, the epigenome of cancer cells is distinguished by aberration of DNA methylation patterns, which include both hypo- and hypermethylation and aberrant regulation of DNA methylation enzymes. In contrast to genetic alterations, which are fixed and are not amenable to therapeutic intervention, pharmacological agents could alter DNA methylation patterns. This raises the prospect that DNA methylation-targeted drugs will reverse cancer growth and metastasis. One of the main challenges however, is to understand the relative role of hypo- and hypermethylation in order to achieve a balance of epigenetic therapeutic agents with positive outcome and reduced adverse effects.
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Affiliation(s)
- Moshe Szyf
- Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, PQ H3G 1Y6, Canada.
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38
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Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C, Hartig R, Schönfeld P, Roessner A, Gali-Muhtasib H. 5-Aza-cytidine is a potent inhibitor of DNA methyltransferase 3a and induces apoptosis in HCT-116 colon cancer cells via Gadd45- and p53-dependent mechanisms. J Pharmacol Exp Ther 2005; 312:525-36. [PMID: 15547111 DOI: 10.1124/jpet.104.074195] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21(WAF1) and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G(2)/M-mediator cyclin B1. Co-incubation with pifithrin-alpha (PFT-alpha), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21(WAF1) expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16(INK4) expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-alpha reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21(WAF1) and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.
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Affiliation(s)
- Regine Schneider-Stock
- Department of Pathology, Division Molecular Genetics, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany.
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Gilbert J, Gore SD, Herman JG, Carducci MA. The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res 2005; 10:4589-96. [PMID: 15269129 DOI: 10.1158/1078-0432.ccr-03-0297] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Methods of gene inactivation include genetic events such as mutations or deletions. Epigenetic changes, heritable traits that are mediated by changes in DNA other than nucleotide sequences, play an important role in gene expression. Two epigenetic events that have been associated with transcriptional silencing include methylation of CpG islands located in gene promoter regions of cancer cells and changes in chromatin conformation involving histone acetylation. Recent evidence demonstrates that these processes form layers of epigenetic silencing. Reversal of these epigenetic processes and up-regulation of genes important to prevent or reverse the malignant phenotype has therefore become a new therapeutic target in cancer treatment.
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Affiliation(s)
- Jill Gilbert
- Division of Oncology and Hematology, Stanley S Scott Cancer Center, Louisiana State University, New Orleans, Louisiana 70112, USA
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40
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Adams AM, Pratt SL, Stice SL. Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells. Mol Reprod Dev 2005; 72:311-9. [PMID: 16078273 DOI: 10.1002/mrd.20357] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
RNA interference (RNAi) has rapidly developed into one of the most widely applied technologies in molecular and cellular research, and although young, is now an essential experimental tool. The versatility of RNAi, especially in mammalian species, lends to its potential applications in a wide array of fields. Without having to genetically manipulate the genome, the ability to selectively reduce the level of a specific transcript using small interfering RNA (siRNA) molecules has great appeal in studying reprogramming issues in somatic cell nuclear transfer (SCNT) embryos. In such embryos, the aberrant expression of the somatic isoform of Dnmt1 (Dnmt1s), the enzyme responsible for maintaining DNA methylation in all somatic cells, has been implicated as one factor in the improper reprogramming of the donor genome. In the present study, the ability to develop a method allowing for the knockdown, or reduction, of Dnmt1s in primary fibroblast cells, like those commonly used as karyoplast donors in SCNT studies, was investigated in primary murine and bovine fibroblast cells as well as in a compromised cell line (NIH/3T3). Two Dnmt1s-specific siRNA candidates were designed and tested. Using optimized conditions, these siRNAs were transiently transfected into the cells with total RNA and nuclear protein being collected. A 56.5% knockdown in Dnmt1s was achieved in the compromised and primary murine cells whereas Dnmt1s was reduced by 15.4% in the primary bovine cells. A reduction in Dnmt1s mRNA did not correspond to a reduction in protein as determined by immunodetection of Western blots. Overall, this study demonstrated the ability of siRNA to knockdown Dnmt1s mRNA in primary fibroblast donor cells. In order to substantially increase the efficiency while decreasing the anomalies seen in SCNT, novel techniques, like the one proposed, are needed to assist the oocyte's ability to reprogram a differentiated genome.
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Affiliation(s)
- A M Adams
- Animal and Dairy Science Department, University of Georgia, 425 River Road, Athens, Georgia 30602, USA
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41
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Santourlidis S, Kimura F, Fischer J, Schulz WA. Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain. Biochem Cell Biol 2004; 82:589-96. [PMID: 15499388 DOI: 10.1139/o04-099] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has been hypothesized to result in aberrant hypermethylation of genes required for cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and human cell lines decreased clonogenicity. By frame-shift and deletion constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino acid domain, which mediates interaction with proliferating cell nuclear antigen (PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies. Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded weaker effects on clonogenicity. Following introduction of the toxic domain, no significant effects on apoptosis, replication, or overall DNA methylation were observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed in cell lines lacking wild-type p53, p21(CIP1), or p16(INK4A). Suppression of clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against carcinogenicity of sustained Dnmt1 overexpression.
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Affiliation(s)
- Simeon Santourlidis
- Urologische Klinik Heinrich Heine Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany.
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42
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Szyf M, Pakneshan P, Rabbani SA. DNA demethylation and cancer: therapeutic implications. Cancer Lett 2004; 211:133-43. [PMID: 15219937 DOI: 10.1016/j.canlet.2004.04.009] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2004] [Accepted: 04/20/2004] [Indexed: 01/12/2023]
Abstract
The epigenome, which is comprised of chromatin and its associated proteins and the patterns of covalent modification of DNA by methylation, sets up and maintains gene expression programs. A hallmark of cancer is a paradoxical aberration of DNA methylation patterns, a global loss of DNA methylation, that coexists with regional hypermethylation of certain genes. The hypermethylation of tumor-suppressor genes has attracted significant attention recently and DNA methylation inhibitors are being tested as potential anticancer agents. However, emerging data suggests that hypomethylation plays a role in activating genes required for metastasis and invasion. It is proposed here that hypermethylation and hypomethylation in cancer are independent processes, which target different programs at different stages in tumorigenesis. Understanding the relative roles of hypomethylation and hypermethylation in cancer has clear implications on the therapeutic use of agents targeting the DNA methylation machinery, which are discussed in this review.
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Affiliation(s)
- Moshe Szyf
- Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, Que., Canada PQ H3G 1Y6.
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43
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Liu ZJ, Zhang XB, Zhang Y, Yang X. Progesterone receptor gene inactivation and CpG island hypermethylation in human leukemia cancer cells. FEBS Lett 2004; 567:327-32. [PMID: 15178346 DOI: 10.1016/j.febslet.2004.04.044] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2004] [Revised: 04/19/2004] [Accepted: 04/19/2004] [Indexed: 10/26/2022]
Abstract
Previous studies showed that progesterone receptor (PR), one of the hormone receptor superfamily, was only connected with the sex-correlated cancers such as breast cancer, endometrial cancer, prostate cancer, etc. This article deals with the PR gene in leukemia. We investigated the methylation status and the expression of the two different PR isoforms, PRA and PRB, in three leukemia cancer cell lines using methylation-specific polymerase chain reaction (MSP-PCR) and reverse transcription-PCR. The correlation of PR methylation and expression together with DNA methyltransferase (DNMT1) was further studied. We found that DNMT1 is required to maintain CpG methylation and aberrant gene silencing of PR gene in human leukemia cancer cells. The activity of 5-aza-2'-deoxycytidine in demethylation and gene reactivation may be through depleting cellular DNMT1 levels. In addition, extensive methylation of PRA and PRB was also observed in leukemia samples. Our results suggest that PR CpG island aberrant hypermethylation could be one molecular and genetic alteration in leukemia.
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Affiliation(s)
- Ze-Jun Liu
- Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
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44
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Suzuki M, Sunaga N, Shames DS, Toyooka S, Gazdar AF, Minna JD. RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. Cancer Res 2004; 64:3137-43. [PMID: 15126351 DOI: 10.1158/0008-5472.can-03-3046] [Citation(s) in RCA: 135] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance methyltransferase gene. Recent studies indicate that inhibiting DNMT1 protein expression may be a useful approach for understanding the role of DNA methylation in tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1 protein expression in NCI-H1299 lung cancer and HCC1954 breast cancer cells. RNA interference-mediated knockdown of DNMT1 protein expression resulted in >80% reduction of promoter methylation in RASSF1A, p16(ink4A), and CDH1 in NCI-H1299; and RASSF1A, p16(ink4A), and HPP1 in HCC1954; and re-expression of p16(ink4A), CDH1, RASSF1A, and SEMA3B in NCI-H1299; and p16(ink4A), RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the tumor cells still were programmed for these epigenetic changes. We saw no effects on soft agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and breast cancer.
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MESH Headings
- Breast Neoplasms/enzymology
- Breast Neoplasms/genetics
- Breast Neoplasms/metabolism
- Carcinoma, Non-Small-Cell Lung/enzymology
- Carcinoma, Non-Small-Cell Lung/genetics
- Carcinoma, Non-Small-Cell Lung/metabolism
- Cell Line, Tumor
- DNA (Cytosine-5-)-Methyltransferase 1
- DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors
- DNA (Cytosine-5-)-Methyltransferases/biosynthesis
- DNA (Cytosine-5-)-Methyltransferases/genetics
- DNA (Cytosine-5-)-Methyltransferases/metabolism
- DNA Methylation
- Gene Expression Regulation, Enzymologic
- Gene Expression Regulation, Neoplastic
- Genes, Tumor Suppressor
- Humans
- Lung Neoplasms/enzymology
- Lung Neoplasms/genetics
- Lung Neoplasms/metabolism
- Promoter Regions, Genetic
- RNA Interference
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- Transfection
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Affiliation(s)
- Makoto Suzuki
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA
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Nelson SM, Ferguson LR, Denny WA. DNA and the chromosome - varied targets for chemotherapy. CELL & CHROMOSOME 2004; 3:2. [PMID: 15157277 PMCID: PMC421739 DOI: 10.1186/1475-9268-3-2] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/04/2004] [Accepted: 05/24/2004] [Indexed: 12/29/2022]
Abstract
The nucleus of the cell serves to maintain, regulate, and replicate the critical genetic information encoded by the genome. Genomic DNA is highly associated with proteins that enable simple nuclear structures such as nucleosomes to form higher-order organisation such as chromatin fibres. The temporal association of regulatory proteins with DNA creates a dynamic environment capable of quickly responding to cellular requirements and distress. The response is often mediated through alterations in the chromatin structure, resulting in changed accessibility of specific DNA sequences that are then recognized by specific proteins. Anti-cancer drugs that target cellular DNA have been used clinically for over four decades, but it is only recently that nuclease specific drugs have been developed to not only target the DNA but also other components of the nuclear structure and its regulation. In this review, we discuss some of the new drugs aimed at primary DNA sequences, DNA secondary structures, and associated proteins, keeping in mind that these agents are not only important from a clinical perspective but also as tools for understanding the nuclear environment in normal and cancer cells.
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Affiliation(s)
- Stephanie M Nelson
- Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 10000, New Zealand
| | - Lynnette R Ferguson
- Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 10000, New Zealand
| | - William A Denny
- Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 10000, New Zealand
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Sun LQ, Lee DW, Zhang Q, Xiao W, Raabe EH, Meeker A, Miao D, Huso DL, Arceci RJ. Growth retardation and premature aging phenotypes in mice with disruption of the SNF2-like gene, PASG. Genes Dev 2004; 18:1035-46. [PMID: 15105378 PMCID: PMC406293 DOI: 10.1101/gad.1176104] [Citation(s) in RCA: 138] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2003] [Accepted: 03/22/2004] [Indexed: 12/21/2022]
Abstract
Imperfect maintenance of genome integrity has been postulated to be an important cause of aging. Here we provide support for this hypothesis by demonstrating that the disruption of PASG (lsh), a SNF2-like factor that facilitates DNA methylation, causes global hypomethylation, developmental growth retardation and a premature aging phenotype. PASG mutant mice display signs of growth retardation and premature aging, including low birth weight, failure to thrive, graying and loss of hair, reduced skin fat deposition, osteoporosis, kyphosis, cachexia, and premature death. Fibroblasts derived from PASG mutant embryos show a replicative senescence phenotype. Both PASG mutant mice and fibroblasts demonstrate a markedly increased expression of senescence-associated tumor suppressor genes, such as p16(INK4a), that is independent of promoter methylation, but, instead, is associated with down-regulation of bmi-1, a negative regulator of p16(INK4a). These studies show that PASG is essential for properly maintaining DNA methylation and gene expression patterns that are required for normal growth and longevity. PASG mutant mice provide a useful model for the study of aging as well as the mechanisms regulating epigenetic patterning during development and postnatal life.
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Affiliation(s)
- Lin-Quan Sun
- Division of Pediatric Oncology, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
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Milutinovic S, Brown SE, Zhuang Q, Szyf M. DNA methyltransferase 1 knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation. J Biol Chem 2004; 279:27915-27. [PMID: 15087453 DOI: 10.1074/jbc.m312823200] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
DNA methyltransferase 1 (DNMT1) catalyzes the post-replication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed.
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Affiliation(s)
- Snezana Milutinovic
- Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada
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48
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Zhu WG, Hileman T, Ke Y, Wang P, Lu S, Duan W, Dai Z, Tong T, Villalona-Calero MA, Plass C, Otterson GA. 5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation. J Biol Chem 2004; 279:15161-6. [PMID: 14722112 DOI: 10.1074/jbc.m311703200] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.
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Affiliation(s)
- Wei-Guo Zhu
- Department of Biochemistry and Molecular Biology, University of Health Science Center, Beijing, China
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Abstract
Vertebrate DNA is modified by methyl moieties at the 5'-position of cytosine rings residing in the di-nucleotide sequence CpG. Approximately 80% of CpG dinucleotide sequences are methylated. The pattern of distribution of methylated CGs is cell-type specific and correlates with gene expression programming and chromatin structure. Three kinds of seemingly contradictory aberrations in DNA methylation are observed in cancer, global hypomethylation, and regional hypermethylation and deregulated level of expression of DNA methyltransferases. It was previously proposed that the DNA methylation machinery is a candidate target for anticancer therapy. Inhibition of hypermethylation was the first therapeutic target. However, recent data suggests that inhibition of DNA methylation might have untoward effects such as induction of genes involved in metastasis. This review discusses the relative role of the three levels of alteration in the DNA methylation in cancer, proposes a unified hypothesis on the relative roles of increased DNA methyltransferase as well as the coexistence of hypo -and hyper- methylation in cancer and its possible implications on anticancer therapy.
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Affiliation(s)
- Moshe Szyf
- Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Promenade, Quebec H3G 1Y6, Montreal, Canada.
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50
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Soejima K, Fang W, Rollins BJ. DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras. Oncogene 2003; 22:4723-33. [PMID: 12879017 DOI: 10.1038/sj.onc.1206510] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2003] [Revised: 02/11/2003] [Accepted: 02/17/2003] [Indexed: 01/03/2023]
Abstract
Transcriptional silencing of tumor suppressor genes in association with DNA methylation contributes to malignant transformation. However, the specific DNA methyltransferases that initiate this process are unknown. Here we show that a de novo DNA methyltransferase, DNMT3b, substantially contributes to the oncogenic phenotype in a lung cancer model. Normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and activated Ras were immortal, formed colonies in soft agar, and expressed DNMT3b. Antisense suppression of DNMT3b prevented soft agar growth. Furthermore, mouse embryo fibroblasts expressing T antigen and Ras formed soft agar colonies and large tumors, but fibroblasts from Dnmt3b(-/-) mice did not grow in soft agar and were much less tumorigenic in vivo. The tumor suppressor genes, FHIT, TSLC1, and RASSF1A were downregulated in transformed NHBE cells, and antisense DNMT3b treatment resulted in re-expression of FHIT and TSLC1. While expression of TSCL1 correlated with methylation of CpG dinucleotides in its promoter region, the expression of FHIT did not, suggesting that DNMT3b may silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin. Regardless of mechanism, our data indicate that DNMT3b plays an important role in transformation.
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Affiliation(s)
- Kenzo Soejima
- Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
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