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

Abstract

// Stéphane Vispé 1 , Arthur Deroide 1 , Emeline Davoine 1 , Cécile Desjobert 1 , Fabrice Lestienne 2 , Lucie Fournier 1 , Natacha Novosad 1 , Sophie Bréand 3 , Jérôme Besse 3 , Florence Busato 4 , Jörg Tost 4 , Luc De Vries 2 , Didier Cussac 2 , Joëlle Riond 1 , Paola B. Arimondo 1 1 Unité 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 Génotypage, CEA-Institut de Génomique, Evry, France Correspondence to: Stéphane Vispé, 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.