A, Aftereffect of vorinostat in the appearance of genes attentive to vorinostat in K562, THP1 and HL60; 1- genes with changed appearance in haematologic malignancies; 2- genes with changed appearance in haematologic malignancies that react to epigenetic therapy; and 3- transcription elements. duplicate. D, Consultant dot blots displaying the percentage of apoptotic K562 and HL60 cells cultured for 72 h in the absence and in the presence of vorinostat. Numbers are percentage of total cells in the respective gates. *p<0.05.(TIF) pone.0053766.s001.tif (1.0M) GUID:?452F3FA4-25CB-44AC-9FE5-B9A2A1CCC014 Figure S2: Effect of vorinostat on terminal erythroid differentiation of K562 cells. K562 cells were treated with vorinostat or vehicle (Control) as indicated. After 4 days, terminal differentiation of K562 was examined by measuring Hb content by ELISA and by microscopy of benzidine (to detect Hb) plus Giemsa stained cells. A, Quantification of hemoglobin content in K562 cells cultured in the presence of vorinostat and vehicle from two different assays, each done in triplicate. Results are expressed as nanograms of Hb per micrograms of total cellular protein SD (n?=?3) in two independent assays. B, benzidine-Giemsa stain of K562 cultured in the absence and in the presence of 2 M vorinostat during 4 days from two independent assays. Similar results were obtained in K562 cells after 3 and 5 days in culture in the absence and presence of vorinostat.(TIF) pone.0053766.s002.tif (905K) GUID:?94DBC519-61FE-4B89-9046-7276931E8CB8 Figure S3: Identification of vorinostat responsive elements in the cFOS and COX2 promoters. A, K562 and HL60 cells were transiently co-transfected with pGL3-bascic vector or reporter constructs containing different DNA sequences of the cFOS promoter cloned into PIK-93 the pGL3-luciferase reporter along with -galactosidase control vector as indicated. 1 h after transfection the cells were treated with 2 M vorinostat or vehicle (Control). Cell lysates were obtained 24 h after and assayed for luciferase and -galactosidase activities. Luciferase activities were normalized to -galactosidase units in the same samples. B, K562 and HL60 cells were transiently co-transfected with pGL3-bascic vector or reporter constructs containing different DNA sequences of the COX2 promoter cloned into the pGL3-luciferase reporter along with -galactosidase control vector as indicated and the rest of the procedure was done as in (A). Results in (A and F11R B) are average fold induction S.D control cells transfected with pGL3-basic PIK-93 of one of three independent assays, PIK-93 done in triplicate, using each reporter plasmid at least from two different clones. Data were analyzed using the ANOVA and the Tukey-Kramer multiple comparison test. *p<0.05.(TIF) pone.0053766.s003.tif (795K) GUID:?C7170036-3889-4D30-9CDD-273D26433B2F Figure S4: Scheme of the proximal promoter regions of IER3, COX2, cFOS, p21, Cyclin G2 and CUL1 genes. Promoter regions of indicated genes were analyzed for the presence of TF binding sites by using the online Transcription Element Search System. The putative binding sites for SP1 and other zinc finger transcription factors present in these sequences are shown. Motifs identical or similar to the GGGAGG motif present in IER3 ?71/?66 promoter region, which is crucial to its basal and vorinostat-mediated expression as by reporter assays, are highlighted.(TIF) pone.0053766.s004.tif (766K) GUID:?54504D0E-32DF-4FCB-808A-10584CEB644E Abstract Background Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action. Methodology/Principal Findings Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33+ cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3) and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1) and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of.