ANTICANCER RESEARCH 31: 2723-2732 (2011) Histone Deacetylase Inhibitors Reverse CpG Methylation by Regulating DNMT1 through ERK Signaling SIBAJI SARKAR, ANA L. ABUJAMRA, JENNY E. LOEW, LORA W. FORMAN, SUSAN P. PERRINE and DOUGLAS V. FALLER Cancer Center, Boston University School of Medicine, Boston, MA, 02118, U.S.A. Abstract. Methylation of CpG repeats in the upstream/ (cytosine-5-)-methyltransferase 1 (DNMT1) levels. Direct promoter regions of genes is an established mechanism of inhibition of ERK activity similarly decreased DNMT1 protein gene silencing in many cell types. DNA methylation results in levels and reversed the basal hypermethylation of the the recruitment of histone deacetylases (HDACs) to promoter promoters and silencing of the RARB2, p21 and p16 tumor regions, thereby repressing expression of genes. General suppressor genes. Suppression of DNMT1 level by siRNA also inhibitors of class I and II HDACs (HDACi), such as sodium reversed methylation of these tumor suppressor genes with butyrate and suberoylanilide hydroxamic acid, suppress the similar kinetics. Collectively, these data demonstrate that growth of prostate cancer cells in vitro and in vivo. In this HDACi, by inhibiting ERK activity, regulate DNMT1 and study, we investigated the mechanism of re-expression of ultimately DNA methylation. These results demonstrate that silenced cell cycle inhibitors and retinoic acid receptor B2 HDACs regulate gene methylation, in addition to the (RARB2). HDACi inhibited cell cycle progression, and established and reciprocal ability of CpG methylation to reversed promoter methylation and silencing of three tumor recruit HDACs to repress transcription. suppressor genes: RARB2 and the cell cycle regulating cyclin- dependent kinase inhibitors p16 and p21. HDACi repressed Methylation of CpG repeats in the upstream/promoter MAP kinase I (ERK) activation and down-regulated DNA regions of genes is an established mechanism of gene silencing and is correlated with the suppression of critical genes, including tumor suppressors, in many types of tumors. The methyl-CpG-binding protein 2 (MeCP2) facilitates This article is freely accessible online. histone deacetylation and DNA methylation-dependent transcriptional silencing. For example, MeCP2 plays an Abbreviations: CpG: Cytosine phosphate guanosine; AZA: 5-aza-2’- important role in neurite extension of PC12 cells by deoxycytidine; HDAC: histone deacetylase; HDACi: histone regulating the expression of key genes involved in deacatylase inhibitors; SB: sodium butyrate; SAHA: suberoylanilide hydroxamic acid; RARB2: retinoic acid receptor beta 2; MAPK1, differentiation (1). Memory formation and synaptic plasticity mitogen-activated protein kinase 1; ERK1: MAP kinase I; AKT1: v- are regulated by DNA methylation and histone acetylation akt murine thymoma viral oncogene homolog 1; DNMTs: DNA (2). Gene silencing by methylation is also prevalent in stem (cytosine-5-)- methyl transferases; DNMT1: DNA (cytosine-5-)- cells, which undergo differentiation while proliferating (3). methyltransferase 1; CDK1: cyclin-dependent kinase 1; MEK1: DNA methylation results in the local recruitment of histone MAP2K1, mitogen-activated protein kinase kinase 1; MEK2: deacetylases (HDACs) to promoter regions with co-localization MAP2K2, mitogen-activated protein kinase kinase 2; p21: CDKN1A, of MeCP2 proteins, eventually inhibiting the binding of RNA cyclin-dependent kinase inhibitor 1A (p21, Cip1); p16: CDKN2A, cyclin-dependent kinase inhibitor 2A; siRNA: small interfering RNA; polymerase II (Pol II) and thereby repressing the expression of MS-PCR: methylation-specific polymerase chain reaction; RT-PCR: genes and their products. Such observations led to the reverse transcriptase-polymerase chain reaction; SDS-PAGE: sodium paradigm that DNA methylation lies ‘upstream’ of histone dodecyl sulphate polyacrylamide gel electrophoresis. acetylation in this pathway of transcriptional repression. While it is known that DNA methyl transferases (DNMTs) Correspondence to: Douglas V. Faller, MD, Ph.D., Cancer Center, produce methylation of CpG repeats in the upstream Boston University School of Medicine, 72 East Concord Street, K- promoter regions of genes, physiological upstream regulators 701, Boston, MA 02118, U.S.A. Tel: +1 6176384173, Fax: +1 6176384176, e-mail: [email protected] of the activity and levels of DNMTs have not been identified. Recent studies suggest that DNA methylation in colon cancer Key Words: Tumor suppressors, cell cycle arrest, DNA N-methyl cells and in NIH 3T3 cells may be regulated in some transferase, ERK. circumstances by ERK (MAP kinase1) activity (4). DNA 0250-7005/2011 $2.00+.40 2723 ANTICANCER RESEARCH 31: 2723-2732 (2011) methylation, and the enzymes which cause methylation, Cell culture. LNCaP cells were obtained directly from ATCC might be modulated directly or indirectly by the extracellular (Manassas, VA, USA), which validates cell line characterizations (ISO milieu through signal transduction pathways, which may Guide 34 accredited), and passaged in the laboratory for fewer than 3 months after receipt. LNCaP prostate cancer cells were grown in include ERK or other cellular kinases. RPMI-1640 media containing 100 IU/ml penicillin, 100 μg/ml General inhibitors of class I and II HDACs, alone or in streptomycin and 50 ml fetal bovine serum. combination with the methylation inhibitor 5-azacitydine, or its congener 5-aza-deoxycytidine (AZA), or other agents are being Cell survival assays. Viable cell enumeration was performed using studied in clinical trials for treatment of diverse types of tumors a trypan blue exclusion assay. The percentage viable cell counts (5). General (class non-specific) HDAC inhibitors (HDACi), were plotted against time of incubation. including sodium butyrate (SB) and suberoylanilide hydroxamic (SAHA), suppress the growth of prostate cancer cells in vitro Methylation-specific PCR (MS-PCR). MS-PCR was performed with bisulfite-treated genomic DNA from treated and untreated cells and in vivo. While the exact mechanisms underlying this growth grown in 100 mm plates. Genomic DNA was isolated using reagents inhibition have not been fully elucidated, it appears that HDACi from Stratagene (La Jolla, CA, USA) according to the induce the expression of several tumor suppressor genes, such manufacturer’s protocol and treated with bisulfite according to a as the cyclin-dependent kinase inhibitors (CDKI) p21 and p16 published protocol (12). MS-PCR was performed with primers (6-9), as well as genes whose products can induce specific for either methylated or bisulfite-modified unmethylated differentiation, such as retinoic acid receptor β2 (RARB2) (10). DNA. The PCR reactions were carried out in a Hybaid PCR Sprint These types of tumor suppressor genes are frequently silenced Thermal Cycler (Hybaid, Franklin, MA, USA). PCR amplifications without genomic DNA were performed as controls. Each PCR in many classes of tumors, including prostate cancer (11). This product was run on a 2% agarose gel, stained with ethidium suggests that exposure to HDACi may influence genes silenced bromide, and visualized under UV illumination. by DNA methylation, either independently of their DNA Total RNA was prepared from pelleted cells using Trizol reagent methylation or by altering their methylation, and thereby (Invitrogen) according to the manufacturer’s protocol. PCR was modulate the growth of tumor cells. performed with cDNA using primers specific for DNMTs 1, 3a, 3b We initiated this work to investigate: (i) whether HDACi and actin, as described below. PCR amplifications without DNA reverse the promoter methylation of silenced genes; (ii) were performed as controls. PCR products were also separated on a 2% agarose gel, stained with ethidium bromide, and visualized whether such promoter demethylation reverses the expression under UV illumination, for quality control. of silenced genes and, if so, (iii) whether HDACi regulate Real-time quantitative PCR was performed with the cDNA DNMT function by a signaling mechanism. We report that prepared as described above for quantitation of RARB2, p21 and p16 exposure to HDACi inhibits the activating phosphorylation transcript expression. The results were normalized with the of ERK, which then causes down-regulation of DNMT1. expression level of β-actin transcripts in each sample. The PCR was Suppression of DNMT1 in turn results in demethylation of performed in triplicate for each sample and the means are presented key tumor suppressor genes, such as RARB2, p21 and p16. with the standard deviations. Primers used for PCR are shown in Tables I-III. Specific repression of DNMT1 by small interfering RNA (siRNA) is sufficient to demethylate and depress expression Cell cycle analyses. For cell cycle analyses, cells were treated with of these tumor suppressor genes, and suppression of ERK SB or SAHA for the indicated times and stained with propidium activity by various means is sufficient to inhibit DNMT1 iodide (PI) as described elsewhere (13) and then analyzed for DNA levels. These studies thus elucidate a pathway in which ERK content by flow cytometry. activity, regulated by HDACi, lies upstream of DNMT1, and DNMT1 suppression, by repression of ERK activity, results siRNA knock-down. DNMT1, MEK1 and MEK2 siRNA Accell Smart in the demethylation of silenced genes. Pools and non-specific siRNAs were obtained from Dharmacon. LNCaP cells were transfected twice at 24 h intervals with siRNAs, according to the protocol provided by the manufacturer. Briefly, Materials