Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4540-4540
Author(s):  
Frank Neumann ◽  
Daniela C. Bruennert ◽  
Anne-Marie Koch ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
K562 Cells ◽  
Differentially Expressed ◽  
Cd34 Cells ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4250-4250
Author(s):  
Daniela Bruennert ◽  
Ingmar Bruns ◽  
Norbert Gattermann ◽  
Ralf Kronenwett ◽  
Rainer Haas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Significant Differential Expression ◽  
Number Of Genes ◽  
In Vitro Treatment

Abstract Abstract 4250 Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures: Gattermann: Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

24 GLOBAL TRANSCRIPT PROFILING OF CLONED BOVINE BLASTOCYSTS USING AFFYMETRIX GENECHIP TECHNOLOGY

2006 ◽  
Vol 18 (2) ◽  
pp. 120
Author(s):  
Z. Beyhan ◽  
P. Ross ◽  
A. Iager ◽  
A. Kocabas ◽  
K. Cunniff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Expression Profiles ◽  
Cell Structure ◽  
Donor Cell ◽  
Lipid Binding ◽  
Differentially Expressed ◽  
Gtp Binding ◽  
Cloned Bovine

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.

The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1352-1352
Author(s):  
Kerstin Schwarz ◽  
Oliver Ottmann ◽  
Annette Romanski ◽  
Anja Vogel ◽  
Jeffrey W. Scott ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Progenitor Cells ◽  
Gene Expression Analysis ◽  
Target Genes ◽  
Notch Pathway ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
20 Nm

Abstract Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC. Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824. Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p<0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.

Gene Expression Profiles of Hydroxyurea Tolerant Cell Lines Identify Genetic Mechanisms That Influence Hydroxyurea Maximum Tolerated Dose

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1005-1005
Author(s):  
Rosa Diaz ◽  
Jonathan M Flanagan ◽  
Thad A Howard ◽  
Russell E. Ware
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Lines ◽  
Expression Profiles ◽  
K562 Cells ◽  
Maximum Tolerated Dose ◽  
False Discovery ◽  
Microarray Expression ◽  
Tolerant Cell

Abstract Abstract 1005 Hydroxyurea has emerged over the past decade as an effective therapeutic agent for patients with sickle cell anemia (SCA). However, drug dosing and hematological responses can be highly variable; both %HbF response and maximum tolerated dose (MTD) vary widely among patients with SCA who receive hydroxyurea treatment. To obtain further insight into the cellular and molecular pathways, as well as genetic factors that might influence the hydroxyurea MTD, K562 erythroleukemia cells were exposed to hydroxyurea in vitro, to create cell lines that were highly drug tolerant to doses ranging from 250μM to 1500μM. Cell lines had dose-response curves that exhibited clear drug tolerance; naïve K562 showed 50% proliferation in the presence of 250μM hydroxyurea, while tolerant cell lines showed >90% proliferation at the same dose as measured by the BrdU Cell Proliferation Assay. In addition, the tolerant lines showed normal and equivalent progression through cell cycle by flow cytometry cell cycle analysis. After 15 weeks of continuous exposure, cells were harvested and mRNA microarray expression profiles were analyzed for naïve K562 (no hydroxyurea exposure) and cell lines tolerant to 500, 1000, or 1500μM hydroxyurea. Gene expression was measured on Affymetrix U133 Plus 2.0 chips. Differential expression between sample groups was determined using ANOVA, and p-values were corrected for multiple testing using the Benjamin-Hochberg false discovery rate (FDR) method to identify genetic profiles and genes consistently increased or decreased compared to naïve K562 cells. Using a threshold of 2-fold change compared to untreated cells and a false discovery rate <5%, a total of 864 genes were significantly altered in hydroxyurea tolerant cells, including 337 genes whose expression consistently correlated with increasing hydroxyurea dose (Pearson correlation p<.001). The PANTHER classification system was used to group genes into categories based on molecular functions. Of the genes that correlated significantly with increasing hydroxyurea dosing (n=337), there were 181 up-regulated genes and 156 down-regulated genes that had molecular functions including catalytic activity, binding, transcription regulator activity and transporter activity. Genes with transporter activity included SLC6A19, ATP6VOD1, ABCG2, ATP6V1B2 and KCNN4. Other genes of interest based on function included RRM2, PLS3, KCNAB2, UBE2A and SRI. Real-time quantitative reverse transcription (RT)-PCR then quantified the expression of 20 candidate genes to verify the accuracy of the microarray expression data. The next steps will include correlation of these findings with clinical data, specifically early reticulocyte mRNA expression and hydroxyurea MTD values obtained from children with SCA enrolled in the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175). These data document that continuous in vitro exposure of K562 cells to hydroxyurea leads to tolerant cell lines that feature substantial changes in gene expression. Altered expression of certain genes present in erythroid cells including RRM2 and membrane transporters represent compensatory changes in response to hydroxyurea exposure, and may help explain the variability in hydroxyurea MTD observed among patients with SCA. Disclosures: Off Label Use: Hydroxyurea is not FDA approved for pediatric sickle cell patients. Howard:Baylor College of Medicine: Employment.

SU11657, a FLT3-Targeted Tyrosine Kinase, Has Pro-Apoptotic Activity on Leukemia Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2797-2797
Author(s):  
Tiziana Grafone ◽  
Laura Ferraretti ◽  
Emanuela Ottaviani ◽  
Manuela Mancini ◽  
Michela Palmisano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Juxtamembrane Region ◽  
Cell Extracts ◽  
Relevant Effect ◽  
Wide Range

Abstract Fms-related tyrosine kinase3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has implicated in its pathogenesis. Constitutive activation of the Flt3 receptor tyrosine kinase, have been linked either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutation in the second tyrosine kinase domain (TKD). To investigate the effect in vitro of SU11657, a new compound FLT3 kinase inhibitors, we analyzed human cell lines from AML patients (MV4-11 and HL60) and blast from patients AML using a wide range of concentrations (1nM-10μM) of this novel agent. In HL-60, FLT3-wt cell line, used as negative control does not show relevant effect after treatment with SU11657. Instead, in MV4-11, FLT3-ITD cell line, we observed a decrease dose-dependent in cell viability after treatment with SU11657. The effects of this compound on cell cycle progression show an accumulation of G1/S phase and an induction of apoptosis at 1-10nM concentration after 24h of treatment. First we observed a dephosphorylation of FLT3 on Tyr591 in whole cell extracts from MV4-11 cells after treatment with SU11657 100nM. We also demonstrated a hypophosphorylation of AKT on Ser473 and a consequently dephosphorylation of BAD on Ser136 at nanomolar concentration. We observed a dephosphorylation of STAT-5 to 100nM of SU11657 at 24h. We evaluated the effects of this new compound in AML primary progenitors that showed FLT3-ITD, FLt3-TKD and FLT3-wt. In the patients with mutation ITD and TKD was evident a modification of cell cycle progression with a decrease in G2/M phase and an increase of subdiploid peak. The effect of SU11657 in patients FLT3-wt was not relevant. Our study thus showed a potential therapeutic usefulness of the drug in treatment of AML. Study of signal transductions and gene profile expression will contribute to further understanding of the drug mechanisms.

scholarly journals Human Wharton’s Jelly Stem Cell Secretions Inhibit Human Leukemic Cell Line K562 in vitro by Inducing Cell Cycle Arrest and Apoptosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Muneerah A. H. Huwaikem ◽  
Gauthaman Kalamegam ◽  
Ghadeer Alrefaei ◽  
Farid Ahmed ◽  
Roaa Kadam ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Inflammatory Cytokine ◽  
K562 Cells ◽  
Cycle Arrest ◽  
Tnf Α ◽  
Ifn Γ

Emerging resistance to the tyrosine kinase inhibitors that target the BCR-ABL1 oncoprotein has prompted research for novel therapeutics against chronic myeloid leukemia (CML). Herein, we evaluated the tumor inhibitory properties of the human Wharton’s jelly stem cells (hWJSCs) co-culture (hWJSC-CC) and their extracts, namely, the hWJSC-conditioned medium (hWJSC-CM; 100%) and hWJSC-lysate (hWJSC-L; 15 μg/ml), on a CML cell line K562 in vitro. The hWJSCs expressed mesenchymal stem cell (MSC)-related cluster of differentiation (CD) markers and demonstrated mesodermal tissue differentiation potential. The cell metabolic activity showed a mean maximal decrease in the K562 cells by 49.12, 41.98, and 68.80% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, respectively, at 72 h. The sub-G1 population in the cell cycle was decreased by 3.2, 4.5, and 3.8% following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L, whereas the G2/M cell population was increased by 13.7 and 12.5% with the hWJSC-CM and hWJSC-L, respectively, at 48 h. Annexin V–allophycocyanin (APC) assay showed an increase in the apoptotic cells by 4.0, 3.9, and 4.5% at 48 h. The expression of pro-apoptotic BAX and CASP3 genes were increased, whereas BIRC5 (Survivin) was decreased compared with the control. The pro-inflammation-related genes, namely, IFN-γ, TNF-α, IL-1β, IL-6, IL-8, and IL-12A, were decreased, whereas the anti-inflammatory genes, namely, IL-4 and IL-10, were increased following treatment with the hWJSC-CC, hWJSC-CM, and hWJSC-L at 48 h. Multiplex bead-based cytokine assay also demonstrated decreases in the pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-6, and IL-12) and an increase in the anti-inflammatory cytokine (IL-10) compared with the control. The pro-inflammatory cytokine IL-8 showed an increase with the hWJSC-CC and decreases with both the hWJSC-CM and the hWJSC-L. The hWJSCs and their extracts inhibited the K562 cells by causing cell cycle arrest and inducing apoptosis via the soluble cellular factors. However, an in vivo evaluation is necessary to unravel the true potential of the hWJSCs and their extracts before its use in CML inhibition.

Gene Expression Profiling of CD34+ Cells in Patients with Myelodysplastic Syndromes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3642-3642
Author(s):  
Andrea Pellagatti ◽  
Mario Cazzola ◽  
Aristoteles Giagounidis ◽  
Janet Perry ◽  
Luca Malcovati ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Hierarchical Clustering ◽  
Myelodysplastic Syndromes ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Good Separation ◽  
Trisomy 8 ◽  
Cd34 Cells

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic malignancies, characterized by blood cytopenias, ineffective hematopoiesis and a hypercellular bone marrow. We have investigated the gene expression profiles of a large group of patients with MDS in order to better understand the molecular pathogenesis of this disorder. The CD34+ cells obtained from 154 MDS patients and 17 healthy individuals were analyzed using Affymetrix U133 Plus2.0 arrays. 38 genes were up-regulated by >2-fold in at least 77 MDS patients, and pathway analysis using these genes showed that the interferon signalling pathway was significantly deregulated (p=0.0006). Indeed IFIT1, the most up-regulated gene (up-regulated in 110 of 154 MDS patients), is an interferon-stimulated gene (ISG). Other ISGs, which mediate growth inhibitory effects of interferon, such as IFITM1, IFI44L and IFIT3, were markedly up-regulated in the majority of MDS patients. Up-regulation of ISGs is a major feature of MDS and may be responsible for some of the hematological characteristics of this disorder, such as peripheral blood cytopenias. We investigated differences in gene expression that could distinguish MDS patients according to their FAB subtype classification (48 patients with RA, 44 patients with RARS and 62 patients with RAEB). Hierarchical clustering performed using the 773 significantly differentially expressed probe sets identified showed that MDS patients with RARS constitute the most homogeneous group, while MDS patients with RA and RAEB show more overlap. RARS gene expression profile was characterized by up-regulation of mitochondrial-related genes and by down-regulation of ABCB7, a gene mutated in the rare inherited X-linked sideroblastic anemia with ataxia (XLSA/A). Moreover, a good separation between the 20 patients with RARS and the 20 patients with RCMD-RS was obtained by hierarchical clustering using the 86 significantly differentially expressed genes between these two WHO subgroups. One of the most significant genes was MFN1, which is essential for mitochondrial fusion and maintenance of mitochondrial morphology. The association of distinct gene expression profiles with specific cytogenetic groups was also determined, and we were able to separate by hierarchical clustering MDS patients with del(5q), patients with −7/del(7q) and patients with trisomy 8. The expression profile of patients with the del(5q) was characterized by down-regulation of genes mapping to chromosome 5q. Genes differentially expressed in patients with −7/del(7q) include LOX and UBE2H, while genes differentially expressed in patients with trisomy 8 include HRSP12 and TPM4. These findings suggest distinct molecular pathogenetic pathways for MDS patients with del(5q), −7/del(7q) and trisomy 8. In order to identify differences in gene expression associated with MDS disease progression, we compared the 48 patients with early MDS (RA) and the 35 patients with advanced MDS (RAEB2). Hierarchical clustering performed using 1081 significantly differentially expressed probe sets resulted in a good separation between MDS patients with RA and patients with RAEB2. LEF1, a regulator of neutrophilic granulopoiesis, was the most significant differentially expressed gene with higher expression levels in patients with RA and decreasing in patients with RAEB2. Other genes showing higher expression levels in patients with RA, decreasing in patients with RAEB2, include CASC5, a cancer susceptibility candidate gene, and RBBP8, a gene that plays a role in DNA-damage-induced cell cycle checkpoint control. Several genes mapping to the cell cycle pathway were significantly deregulated between early and advanced MDS. This study provides new important insights into the pathophysiology of MDS and represents a first step towards determining pathway signatures in MDS as a guide to targeted therapies.

A Distinct Expression of Various Gene Subsets in CD34+ Cells Obtained From Patients with Early and Advanced Myelodysplastic Syndrome.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3795-3795
Author(s):  
Monika Belickova ◽  
Jaroslav Cermak ◽  
Alzbeta Vasikova ◽  
Eva Budinska
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Differentially Expressed Genes ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Abnormal Development ◽  
Statistical Hypothesis ◽  
P Value ◽  
Cd34 Cells

Abstract Abstract 3795 Poster Board III-731 Gene expression profiles of CD34+ cells were compared between a cohort of 51 patients with MDS or AML from MDS and 7 healthy controls. The patients were classified according to the WHO criteria as follows: 5q- syndrome (n=7), RA (n=3), RARS (n=2), RCMD (n=10), RAEB-1 (n=7), RAEB-2 (n=15), and AML with MLD (multilineage dysplasia) (n=7). HumanRef-8 v2 Expression Bead Chips (Illumina) were used to generate expression profiles of the samples for >22,000 transcripts. The raw data were normalized data with the R software, lumi package. Normalized data were filtered by detection p-value <0.01, resulting in total number of 9811genes. To identify differentially expressed genes we performed two parallel statistical hypothesis testings: Analysis of Variance (ANOVA) together with Tukey test and empirical bayesian thresholding correction for multiple testing problem; and Significance Analysis of Microarrays (SAM). The results were confirmed by real-time quantitative PCR for six genes (TaqMan Gene Expression Assays). Hierarchical clustering of significantly differentially expressed genes clearly separated patients and controls, 5q-syndrome and RAEB-1 as a separate entities confirming usefulness of WHO classification subgroups. The most up-regulated genes in all patients included HBG2, HBG1, CYBRD1, HSPA1B, ANGPT1, and MYC. We assume that expression changes in globin genes, both fetal and adult globins (HBG2, HBG1 and HBA1, HBB) may play role not only in dysregulation of erythropoiesis but also in the disease progression or leukemic transformation of MDS. Among the most down-regulated genes, 13 genes related to B-lymphopoiesis (e.g. POU2AF1, VPREB1, VPREB3, CD79A, EBF1, LEF1, BCL3, IRF8 & IRF4) were detected, suggesting the abnormal development of B-cell progenitors in all MDS patients. Some of these genes (e.g. VPREB3, LEF1) showed decreasing trend in expression level from early to advanced MDS with the lowest expression in AML with MLD. Patients with advanced MDS had significantly decreased expression of genes involved in in the mitotic cell cycle, DNA replication, and chromosome segregation compared to early MDS where these gene subsets were up-regulated. The DAVID database also identified de-regulation in the cell cycle pathway through its 7 genes (CDC25C, CDC7, CDC20, ORC1L, CCNB2, BUB1, & CCNA2). On the other hand, advanced MDS patients showed significant up-regulation of proto-oncogenes (BMI1, MERTK) and genes related to angiogenesis (ANGPT1), anti-apoptosis (VNN1). The results confirm on molecular basis that increased cell proliferation and resistance to apoptosis together with a loss of cell cycle control, damaged DNA repair and altered immune response may play an important role in the expansion of malignant clone in MDS patients. The study was supported by Grant NR-9235 obtained from the Ministry of Health, Czech Republic. Disclosures: No relevant conflicts of interest to declare.

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