Description
Accession Number: GSE19610
Platform:
GPL96: [HG-U133A] Affymetrix Human Genome U133A Array
Organism: Homo sapiens
Published on 2009-12-23
Summary:
Epigenetic mechanisms contribute to deregulated gene expression of hematopoietic progenitors in Myelodysplastic Syndromes (MDS). Hypomethylating agents are able to improve peripheral cytopenias in MDS patients. To identify critical gene expression changes induced by hypomethylating agents, we analyzed gene expression profiling (GEP) of myelodysplastic and normal CD34+ hematopoietic stem cells treated in vitro with or without decitabine. Four MDS and two untreated early stage Hodgkin’s lymphomas were analyzed for GEP. Mock treated CD34+ stem cells segregate according to diagnosis and karyotype. After decitabine treatment, gene expression changes were more consistent on MDS CD34+ cells with abnormal kayotype. Comparing decitabine-induced genes with those found down-regulated in mock-treated MDS cells, we identified a list of candidate tumor suppressor genes in MDS. By real-time RT-PCR we confirmed expression changes for three selected genes CD9, CXCR4 and GATA2 in 12 MDS patients and 4 controls. CD9 was widely repressed in most MDS CD34+ cell samples, although similar levels of methylation were found in both normal and MDS total bone marrows. CXCR4 promoter methylation was absent in total bone marrows from 36 MDS patients. In conclusion, changes in gene expression changes induced by hypomethylating treatment are more pronounced in CD34+ cells from abnormal karyotype.
Overall Design:
Bone marrow mononuclear cells were obtained by Ficoll gradient centrifugation from 4 patients with intermediate-2/high risk Myelodysplastic Syndromes (MDS) (two with normal and two with abnormal karyotype) and from two patients with untreated early-stage Hodgkin’s lymphoma (HL). CD34+ cells were freshly isolated using immunomagnetic beads (Minimacs, Milteny Biotec GmbH, Germany), according to the manufacturer’s instructions. CD34+ cells were cultured in 24-well-plates with a pool of growth factors, including 10 ng/ml each of Stem Cell Factor, Flt3 ligand, IL3 and Thrombopoietin (Sigma Aldrich), for 24 hours. Decitabine (Sigma Aldrich) was then added to a final concentration of 1 µM for the following 72 hours, while the corresponding amount of solvent was added for the mock treated plates. Three biological replicates were prepared for each patient (for both decitabine- and mock-treated). At the end of the treatment cells were harvested and total RNA was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The GeneChip® Two-Cycle Target Labeling and Control Reagents kit (Affymetrix) was used to amplify and label RNA, according to the manufacturer’s instructions. Biotinilated cRNA were then hybridized on Affymetrix GeneChips HG-U133A. We used 5 arrays for each patient: three replicates for decitabine treated CD34+ cells and two replicates for mock treated cells. Data were analyzed by the software Bioconductor (www.bioconductor.org and www.bioinformatica.unito.it) through the program RGui version 2.5.0, package oneChannelGUI. Data were then normalized by GCRMA. The Bayesian method Linear Model Analysis of Microarray (Limma) was used to identify differentially expressed genes, by sorting data with p value 1. Contrasts were analyzed for diagnosis (MDS vs HL), karyotype (normal vs abnormal) and treatment (decitabine vs mock).
Contact:
Name: Francesco D'Alò
Organization: Università Cattolica del Sacro Cuore
Laboratory: Molecular Biology
Deparment: Hematology
Address: Largo A. Gemelli 8 Roma 00168 Italy
Email: [email protected]
Phone: +393497894529
Organization: Affymetrix, Inc.
Address: Santa Clara CA 95051 USA
Email: [email protected], [email protected]
Phone: 888-362-2447
Web-Link: http://www.affymetrix.com/index.affx
Platform:
GPL96: [HG-U133A] Affymetrix Human Genome U133A Array
Organism: Homo sapiens
Published on 2009-12-23
Summary:
Epigenetic mechanisms contribute to deregulated gene expression of hematopoietic progenitors in Myelodysplastic Syndromes (MDS). Hypomethylating agents are able to improve peripheral cytopenias in MDS patients. To identify critical gene expression changes induced by hypomethylating agents, we analyzed gene expression profiling (GEP) of myelodysplastic and normal CD34+ hematopoietic stem cells treated in vitro with or without decitabine. Four MDS and two untreated early stage Hodgkin’s lymphomas were analyzed for GEP. Mock treated CD34+ stem cells segregate according to diagnosis and karyotype. After decitabine treatment, gene expression changes were more consistent on MDS CD34+ cells with abnormal kayotype. Comparing decitabine-induced genes with those found down-regulated in mock-treated MDS cells, we identified a list of candidate tumor suppressor genes in MDS. By real-time RT-PCR we confirmed expression changes for three selected genes CD9, CXCR4 and GATA2 in 12 MDS patients and 4 controls. CD9 was widely repressed in most MDS CD34+ cell samples, although similar levels of methylation were found in both normal and MDS total bone marrows. CXCR4 promoter methylation was absent in total bone marrows from 36 MDS patients. In conclusion, changes in gene expression changes induced by hypomethylating treatment are more pronounced in CD34+ cells from abnormal karyotype.
Overall Design:
Bone marrow mononuclear cells were obtained by Ficoll gradient centrifugation from 4 patients with intermediate-2/high risk Myelodysplastic Syndromes (MDS) (two with normal and two with abnormal karyotype) and from two patients with untreated early-stage Hodgkin’s lymphoma (HL). CD34+ cells were freshly isolated using immunomagnetic beads (Minimacs, Milteny Biotec GmbH, Germany), according to the manufacturer’s instructions. CD34+ cells were cultured in 24-well-plates with a pool of growth factors, including 10 ng/ml each of Stem Cell Factor, Flt3 ligand, IL3 and Thrombopoietin (Sigma Aldrich), for 24 hours. Decitabine (Sigma Aldrich) was then added to a final concentration of 1 µM for the following 72 hours, while the corresponding amount of solvent was added for the mock treated plates. Three biological replicates were prepared for each patient (for both decitabine- and mock-treated). At the end of the treatment cells were harvested and total RNA was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The GeneChip® Two-Cycle Target Labeling and Control Reagents kit (Affymetrix) was used to amplify and label RNA, according to the manufacturer’s instructions. Biotinilated cRNA were then hybridized on Affymetrix GeneChips HG-U133A. We used 5 arrays for each patient: three replicates for decitabine treated CD34+ cells and two replicates for mock treated cells. Data were analyzed by the software Bioconductor (www.bioconductor.org and www.bioinformatica.unito.it) through the program RGui version 2.5.0, package oneChannelGUI. Data were then normalized by GCRMA. The Bayesian method Linear Model Analysis of Microarray (Limma) was used to identify differentially expressed genes, by sorting data with p value 1. Contrasts were analyzed for diagnosis (MDS vs HL), karyotype (normal vs abnormal) and treatment (decitabine vs mock).
Contact:
Name: Francesco D'Alò
Organization: Università Cattolica del Sacro Cuore
Laboratory: Molecular Biology
Deparment: Hematology
Address: Largo A. Gemelli 8 Roma 00168 Italy
Email: [email protected]
Phone: +393497894529
Organization: Affymetrix, Inc.
Address: Santa Clara CA 95051 USA
Email: [email protected], [email protected]
Phone: 888-362-2447
Web-Link: http://www.affymetrix.com/index.affx
| Dati resi disponibili | 22 dic 2009 |
|---|---|
| Editore | Gene Expression Omnibus |