1. Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells
- Author
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Joëlle Riond, Didier Cussac, Lucie Fournier, Florence Busato, Jérome Besse, Cécile Desjobert, Jörg Tost, Sophie Bréand, Stéphane Vispé, Emeline Davoine, Natacha Novosad, Fabrice Lestienne, Luc De Vries, Arthur Deroide, Paola B. Arimondo, Pharmacochimie de la Régulation Epigénétique du Cancer (ETaC), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-PIERRE FABRE, Centre de Recherche Pierre Fabre (Centre de R&D Pierre Fabre), PIERRE FABRE, Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by Centre National de la Recherche Scientifique (CNRS) [ATIP to P.B.A.], and Région Midi Pyrenées [Equipe d'Excellence and FEDER CNRS/Région Midi Pyrenées to P.B.A] and Fondation InNaBioSanté [to P.B.A.]., The authors thank Chantal Etievant and Jean-Marc Gregoire for helpful discussions., and PIERRE FABRE-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
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[SDV]Life Sciences [q-bio] ,DNMT ,MESH: G2 Phase Cell Cycle Checkpoints ,DNA Methyltransferase 3A ,Histones ,MESH: DNA Methylation ,MESH: RNA, Small Interfering ,DNA (Cytosine-5-)-Methyltransferases ,Cancer epigenetics ,Phosphorylation ,RNA, Small Interfering ,Promoter Regions, Genetic ,MESH: CpG Islands ,MESH: Histones ,DNA methylation ,biology ,leukemia ,Nuclear Proteins ,3. Good health ,DNA-Binding Proteins ,G2 Phase Cell Cycle Checkpoints ,Oncology ,embryonic structures ,Azacitidine ,MESH: Tumor Protein p73 ,RNA Interference ,Research Paper ,5-aza-2′-deoxycytidine ,DNA (Cytosine-5-)-Methyltransferase 1 ,MESH: DNA (Cytosine-5-)-Methyltransferases ,MESH: Cell Line, Tumor ,DNA repair ,DNA damage ,MESH: RNA Interference ,MESH: Azacitidine ,Decitabine ,DNA methyltransferase ,5-aza-2’-deoxycytidine ,Cell Line, Tumor ,MESH: Cell Proliferation ,MESH: Leukemia ,MESH: Promoter Regions, Genetic ,Humans ,[CHIM]Chemical Sciences ,MESH: Tumor Suppressor Proteins ,Epigenetics ,5-aza-2'-deoxycytidine ,Cell Proliferation ,MESH: DNA (Cytosine-5-)-Methyltransferase 1 ,MESH: DNA Damage ,MESH: Humans ,MESH: Phosphorylation ,MESH: Decitabine ,Tumor Suppressor Proteins ,Tumor Protein p73 ,Molecular biology ,Proliferating cell nuclear antigen ,DNA demethylation ,biology.protein ,Cancer research ,CpG Islands ,MESH: Nuclear Proteins ,MESH: DNA-Binding Proteins - Abstract
// Stephane Vispe 1 , Arthur Deroide 1 , Emeline Davoine 1 , Cecile Desjobert 1 , Fabrice Lestienne 2 , Lucie Fournier 1 , Natacha Novosad 1 , Sophie Breand 3 , Jerome Besse 3 , Florence Busato 4 , Jorg Tost 4 , Luc De Vries 2 , Didier Cussac 2 , Joelle Riond 1 , Paola B. Arimondo 1 1 Unite de Service et de Recherche n°3388 CNRS-Pierre Fabre, ETaC Epigenetic Targeting of Cancer, CRDPF, Toulouse, France 2 Molecular and Cellular Biology Department, Centre de Recherche Pierre Fabre, Castres, France 3 Informatique de Recherche (Bioinformatics and Statistics), Centre de Recherche Pierre Fabre, Castres, France 4 Laboratory for Epigenetics and Environment, Centre National de Genotypage, CEA-Institut de Genomique, Evry, France Correspondence to: Stephane Vispe, e-mail: stephane.vispe@pierre-fabre.com Paola B. Arimondo, e-mail: paola.arimondo@etac.cnrs.fr Keywords: leukemia, DNA methylation, DNMT, 5-aza-2’-deoxycytidine, DNA damage Received: December 28, 2014 Accepted: February 08, 2015 Published: March 20, 2015 ABSTRACT 5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.
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- 2015