Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2’-deoxycytidine in human leukemic KG1 cells
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Stéphane Vispé1, Arthur Deroide1, Emeline Davoine1, Cécile Desjobert1, Fabrice Lestienne2, Lucie Fournier1, Natacha Novosad1, Sophie Bréand3, Jérôme Besse3, Florence Busato4, Jörg Tost4, Luc De Vries2, Didier Cussac2, Joëlle Riond1, Paola B. Arimondo1
1Unité de Service et de Recherche n°3388 CNRS-Pierre Fabre, ETaC Epigenetic Targeting of Cancer, CRDPF, Toulouse, France
2Molecular and Cellular Biology Department, Centre de Recherche Pierre Fabre, Castres, France
3Informatique de Recherche (Bioinformatics and Statistics), Centre de Recherche Pierre Fabre, Castres, France
4Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France
Stéphane Vispé, e-mail: [email protected]
Paola B. Arimondo, e-mail: [email protected]
Keywords: leukemia, DNA methylation, DNMT, 5-aza-2’-deoxycytidine, DNA damage
Received: December 28, 2014 Accepted: February 08, 2015 Published: March 20, 2015
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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