title: DNA methylation in ATRA-treated leukemia cell lines lacking a PML-RAR chromosome translocation.
abstract: A deficient retinoic acid signaling has been suggested to be an important cause of the clinical inefficacy of all-trans retinoic acid (ATRA) therapy in non-promyelocytic (non-PML) forms of acute myeloid leukemia (AML). The general aim of the present work was to explore novel ways to take advantage of the anti-leukemic potential of ATRA, and, specifically, to search for a synergism between ATRA and epigenetic drugs. Because previous reports have found no major influence of ATRA on DNA methylation, we investigated whether ATRA-mediated differentiation of the U937 and HL-60 AML cell lines, both lacking a PML-retinoic acid receptor (RAR) fusion product, is accompanied by early-appearing and weak changes in CpG methylation. We report that in HL-60 cells, by using a highly quantitative analysis of a set of genes found to be abnormally expressed in AML, polymerase chain reaction (PCR)-amplified p16 gene promoter molecules (each with 15 CpG sites), exhibited a CpG methylation level of 0-4% in untreated cells, which increased to 4-21% after treatment with ATRA for seven days. In contrast to HL-60 cells, U937 cells exhibited a very high CpG methylation level in p16, and ATRA did not influence the promoter methylation of this gene. In the total CCGG sites of the genome, analysed using a methylation-sensitive restriction enzyme, CpG methylation was significantly lower in ATRA-treated HL-60 (p<0.01) and U937 cells (p<0.05) than in controls. Taken together, our findings show that ATRA can influence DNA methylation, and suggest that future research should investigate whether epigenetic modulation may evoke a clinical effect of ATRA in leukemia.
authors.affiliation: Department of Laboratory Medicine, Skane University Hospital, Lund University, 205 02 Malmö, Sweden.
authors.forename: Regina
authors.initials: R
authors.lastname: Miftakhova
authors.forename: Tove
authors.initials: T
authors.lastname: Sandberg
authors.forename: Andreas
authors.initials: A
authors.lastname: Hedblom
authors.forename: Tatyana
authors.initials: T
authors.lastname: Nevzorova
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Persson
authors.forename: Anders
authors.initials: A
authors.lastname: Bredberg
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D018899:CpG Islands; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019175:DNA Methylation / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics; D015514:Oncogene Proteins, Fusion; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: D000970:Antineoplastic Agents; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin