1626 REGULATORY SEQUENCES OF THE ATP-BINDING CASSETTE TRANSPORTER A1 (ABCA1) GENE ARE HYPERMETHYLATED IN PROSTATE CANCER RESULTING IN LOSS OF GENE EXPRESSION

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111626 REGULATORY SEQUENCES OF THE ATP-BINDING CASSETTE TRANSPORTER A1 (ABCA1) GENE ARE HYPERMETHYLATED IN PROSTATE CANCER RESULTING IN LOSS OF GENE EXPRESSION Byron Lee, Cristina Magi-Galluzzi, Eric Klein, and Angela Ting Byron LeeByron Lee Cleveland, OH More articles by this author , Cristina Magi-GalluzziCristina Magi-Galluzzi Cleveland, OH More articles by this author , Eric KleinEric Klein Cleveland, OH More articles by this author , and Angela TingAngela Ting Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1734AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cholesterol homeostasis is altered in prostate cancer resulting in increased levels of cholesteryl esters and free cholesterol, but the mechanism behind these perturbances is not complete understood. We examine the role of DNA methylation in the regulation of ABCA1 expression. METHODS Genome wide methylation analysis using MBD2-isolated Genome Sequencing (MiGS) was performed on the normal prostate epithelial cell line PrEC, the prostate cancer cell lines LNCaP and DU 145, 3 benign prostate specimens obtained from radical cystoprostatectomy, and 6 prostate cancer specimens obtained from radical prostatectomy. Bisulfite genomic sequencing was performed on the ABCA1 CpG island in PrEC and LNCaP to verify the methylation patterns discovered by MiGS. Real time RT-PCR was performed to assess ABCA1 mRNA levels in PrEC and LNCaP. LNCaP was treated with 5-aza-2'-deoxycytidine (5-aza), the LXR agonist T0901317, or a combination of the two drugs; and ABCA1 CpG island methylation was assessed by methylation-specific PCR (MSP) and gene expression was assessed by real time RT-PCR. RESULTS MiGS analysis revealed that ABCA1 CpG island methylation was present in LNCaP and 6 prostate cancer specimens but not PrEC, DU 145, or 3 benign prostate specimens. Bisulfite genomic sequencing primers were used to amplify fragments from the CpG island spanning –404 to +1,301 of the ABCA1 gene. LNCaP showed dense methylation in the entire region while PrEC showed only minimal methylation from –236 to –404. Real time RT-PCR revealed that there was 10-fold more ABCA1 mRNA in PrEC when compared with LNCaP. Treatment of LNCaP with 10μM T0901317 for 24 hours increased ABCA1 mRNA to 14 fold above untreated LNCaP without detectable changes in DNA methylation. Treatment with 5μM 5-aza for 2 weeks increased ABCA1 mRNA to 50 fold above control and resulted in loss of DNA methylation in the ABCA1 CpG island as detected by MSP. The combination of 5μM 5-aza and 10μM T0901317 increased ABCA1 mRNA expression in LNCaP to 320 fold above control. CONCLUSIONS ABCA1 is hypermethylated in a subset of prostate cancers, but not normal prostate, resulting in loss of gene expression. Therapeutic strategies aimed at reducing intracellular cholesterol content solely by inhibiting HMG-CoA reductase may be hindered by this acquired deficiency in cholesterol export. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e652 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Byron Lee Cleveland, OH More articles by this author Cristina Magi-Galluzzi Cleveland, OH More articles by this author Eric Klein Cleveland, OH More articles by this author Angela Ting Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...