@article{d3041ea9a0624dc1860bc5f1cee80f46,
title = "Five-aza-2′-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells",
abstract = "DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.",
author = "Takayuki Tsujioka and Akira Yokoi and Yoshitaro Itano and Kentaro Takahashi and Mamoru Ouchida and Shuichiro Okamoto and Toshinori Kondo and Suemori, {Shin Ichiro} and Yumi Tohyama and Kaoru Tohyama",
note = "Funding Information: Quantitative real-time reverse transcription PCR. Quantitative real-time reverse transcription PCR (q-PCR) was performed with Applied Biosystems StepOne PlusTM Real time PCR system (Life Technologies Japan Ltd, Tokyo). Total cellular RNA (3–5μg) was used to synthesize cDNA by using Ready-To-Go T primed First-Strand Kit (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK) in a final volume of 33μl. Five μl of 1:10 diluted cDNA reactions was used as input for each of the real-time quantitative PCR reactions by using SYBR Premix Ex TaqTM (Takara Bio Inc, Shiga, Japan). Initial denaturation at 95 °C for 20 s was followed by 40 cycles of a denaturation step at 95 °C for 3 s and an annealing and extension step at 60 °C for 30 s. Primers were designed by using Primer 3 v 4.0.0 software (Initial development of Primer3 was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute). Since human CH25H gene spans one exon, we used total RNA treated with DNase I (Qiagen, Tokyo, Japan) so that the experimental results are not compromised through genomic DNA contamination33. Funding Information: Support and Financial Disclosure Declaration. Tohyama K received research funding from Eisai Co., Ltd. The other authors have no competing financial interests to declare Funding Information: This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; and in part by a Kawasaki Medical School Project Grant and Kurozumi Medical Foundation Grant. The authors thank Prof. Takashi Sugihara and Prof. Hideho Wada, a Division of Hematology, Department of Internal Medicine, Kawasaki Medical School for providing the patient samples, and Ms. Aki Kuyama and Ms. Asami Matsuo for editorial assistance. Publisher Copyright: {\textcopyright} 2015, Nature Publishing Group. All rights reserved.",
year = "2015",
month = nov,
day = "18",
doi = "10.1038/srep16709",
language = "English",
volume = "5",
journal = "Scientific reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
}