ated B cells, which retain their ability to divide in conjunction with a differentiated phenotype. In acute leukemia cells, in which an early block in differentiation leads to malignancy, LBH589 induces differentiation [25]. It is therefore of interest to note that the reduction of growth in MM by LBH589 is the result of cell cycle arrest [39], fulfilling at least one of the criteria of differentiation. Additional studies are required to dissect the functional role of the repressed gene signature in maintaining proliferation in MM, and the biological consequences of its reactivation. The existence of the silenced gene expression profile in MGUS, recently proposed to precede MM in majority of the cases [46,47,48], suggests that aberrant Polycomb-mediated gene silencing might be an early event during MM development. Although a small subpopulation of clonogenic CD138 negative B cells are suggested to function as cancer stem cells in MM [49], they are unlikely to have an impact on the underexpressed gene profile, when analyzing the tumor bulk. Instead, the identification of the Polycomb-silenced gene profile in CD138 positive patient samples and cell lines implies that this is a general feature shared by a large proportion of the MM cells. The question, however, remains if MM cells have acquired the Polycomb gene repression signature de novo in the late stages of plasma cell differentiation, or if it is a remnant from earlier stages of normal B-cell development, which remains preserved in the MM cells. Though, currently we cannot rule out any of these possibilities, our data argues that maintaining a stemness feature in the plasma cell tumor is important in order to sustain survival and self-renewal of MM. Materials and Methods Ethics Statements The study involving human biopsy samples was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committees of Stockholm and Uppsala (Dnr 2004:M-332). Patients gave written informed consent for the sample collection. In the animal studies animals had free access to food and water and they were housed and treated following the conditions approved by the Ethical Committee for Animal Experiments, VUB (license nr LA1230281). Bioinformatic mining Publically available microarray data were explored using the Oncomine database [21]. Briefly, microarray data were log- transformed, median centered per array, and standard deviation normalized to one per array. Genes underexpressed in MM datasets were compared to literature defined concepts in Oncomine, using the built in t-statistics. Gene-set enrichment analysis was performed using the GSEA v2.05 software from Broad Institute as previously described [50]. The five selected target genes (CIITA, CXCL12, GATA2, CDH6 and ICSBP/IRF8) were compared with the Mayo clinic data set from Gene Expression Omnibus (GSE6477). The Mayo clinic dataset contains 15 normal bone marrows that were compared to 147 different grades of MM patients. Purification of MM cells from patient material and MM cell lines Heparinised bone marrow samples were obtained from 4 newly diagnosed MM patients. Mononuclear cells were separated by Ficoll- Paque Plus density sedimentation (Amersham Biosciences, Little Chalfont, UK) and were subjected to CD138 immunomagnetic purification according to the manufacturer’s protocol (Miltenyi Biotech, Paris, France). The enriched fraction gave a purity of greater than 95%, determined by May-Gru¨nwald-Giemsa staining. Polycomb Silencing in MM The MM cell lines RPMI 8226 [51] and U-266-1984 [52] (source ATCC and Kenneth Nilsson, Uppsala University, respectively) were maintained in RPMI-1640 AQmediaTM (SigmaH) supple- mented with 10% fetal bovine serum (FBS, SigmaH) and antibiotics (penicillin 100 U/ml and streptomycin 50 mg/ml; SigmaH) at 37uC in a humidified 5% CO2 in-air atmosphere. Exponentially growing cells were seeded at 26105 cells/ml (RPMI 8226) or 46105 (U-266- 1984) and incubated overnight before addition of reagents. Reagents LBH589 (Panobinostat, Novartis Pharmaceuticals Inc.) was dissolved in dimethyl sulfoxide (DMSO) and DZNep (National Cancer Institute, Bethesda, MD, USA) was dissolved in dH2O. Both chemical inhibitors were used at the indicated concentra- tions. The concentration of solvent (DMSO or dH2O) in each experiment (,0.1%) did not alter the growth and survival of the MM cells (data not shown). Chromatin immunoprecipitation (ChIP) Proteins were cross-linked to DNA using 1% formaldehyde in serum-free medium for 10 min in room temperature. Cross- linking was inhibited by 125 mM glycine. Cells were washed in ice-cold phosphate-buffered saline (PBS), treated with cell lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 and protease inhibitors) for 10 min on ice and cell nuclei were collected at 2500 rpm for 5 min. Nuclei were lysed in RIPA buffer (MOPS (free acid), 0.5 M EDTA pH 8.0, 5N NaCl (to adjust pH to 7.0– 7.2), 1% SDS; 10% NP-40, 10% DOC and protease inhibitors) for 10 min on ice and sonicated 3615 min (30 sec ON/30 sec OFF) at ultrasonic wave output power 320 W in BioruptorH (Diagenode, Lie`ge, Belgium). Chromatin was collected at 14 000 rpm for 15 min and divided in several fractions. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07- 449) and IgG (Negative control, OneDay ChIP KitTMDiagenode). Chromatin immunoprecipitation was performed using OneDay ChIP KitTM (Diagenode, Lie`ge, Belgium) according to the manufacturer’s protocol. Precipitated DNA was analyzed by Real Time-qPCR using PlatinumH SYBRH Green qPCR SuperMix UDG with Rox (Invitrogen, Carlsbad, CA) and 0.3 mM of each forward and reverse primers. Primer sequences are given in supplementary data (Table S2). The PCR conditions were: 95uC for 2 min followed by 40 cycles of 95uC for 0:30 min and 60uC for 1 min. The run and analysis were performed using Mx3005P instrument and software (Stratagene). RNA extraction, cDNA synthesis and Quantitative Real- Time RT-PCR Total RNA was extracted using TRIzolH (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Reverse transcrip- tion using random primers (Invitrogen) was performed on 1–2 mg of total RNA using SuperScriptTM III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. Each independent quantitative real-time reverse transcriptase PCR (qRT-PCR) reaction contained 5 ng of cDNA, TaqManH Gene Expression Master Mix (Applied Biosystems, Foster City, CA) and TaqManH Gene Expression Assays (Applied Biosystems) according to manufacturer’s recommendations. The run and analysis were performed using Mx3005P instrument and software (Stratagene). Western Blot At the indicated time points, RPMI 8226 and U-266-1984 cells were harvested, washed with ice-cold PBS and resuspended in lysis PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11483