Distinct DNA Methylation Patterns of HOX Genes in Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2238-2238
Author(s):  
Amanda J. Saraf ◽  
Samantha F. Lau ◽  
Rong He ◽  
Jean-Pierre J. Issa ◽  
Jaroslav Jelinek
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Blood Cells ◽  
Hox Genes ◽  
Cpg Islands ◽  
Leukemic Cell ◽  
Epigenetic Silencing ◽  
Myelogenous Leukemia ◽  
Leukemic Cell Lines ◽  
Normal Controls

Abstract The HOX subset of homeobox gene family comprises 39 genes organized in 4 clusters A-D on 4 different chromosomes and in 13 paralog groups defined across the clusters. HOX genes are important regulators of development and tissue differentiation. Dysregulated expression disturbs hematopoiesis and HOX genes are frequent partners in chromosomal translocations involved in leukemia. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing of affected genes. Methylation pattern is faithfully copied during cell replication to subsequent cell generations and hypermethylation thus represents a permanent mark functionally equivalent to a loss-of-function mutation. Epigenetic silencing of HOX genes may disrupt normal development of blood cells and thus be involved in the development of leukemia, as was recently reported for HOXA5. We performed a comprehensive DNA methylation analysis of 22 HOX genes with CpG islands in promoter to exon 1 regions: HOXA1, A4, A5, A7, A9, A10, A11; B1, B4, B5, B7, B8, B9, B13; C4, C5, C8, C9, C10, C13; D1 and D10 in 16 leukemic cell lines, 24 samples from patients with acute myelogenous leukemia (AML), 20 samples from patients with acute lymphoblastic leukemia (ALL), and 15 control samples of normal blood cells. We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Overall, lymphoblastic leukemic cell lines (TALL, Raji, ALL1, JTAg, CEM, BJAB, Jurkat) showed the highest degree of HOX genes methylation (average 60%), followed by myeloid leukemic cell lines (HL60, KG1a, ML1, HEL, TF-1, OCI-AML3, K562, TF1i, KG1, average methylation 40%), p=0.000. Analysis of ALL and AML patient samples revealed significant differences in methylation levels of several HOX genes. Consistently with their reported overexpression in leukemia, HOX A9, B4, and also HOXB5 and HOXD10 were seldom methylated in AML patients (2/24, 0/24, 1/12, 7/24, respectively), while hypermethylation over a 10% cutoff value was observed frequently in ALL patients (10/20, 6/19, 6/17, 9/19, respectively). The differences in methylation were statistically significant (p<0.05). On the other hand, HOXA4 showed significantly higher methylation densities in AML (median 74%, range 35–95%) and ALL (median 49%, range 12–91%) than in normal controls (median 34%, range 17–59%), p<0.01. HOXC4 was more frequently methylated in normal controls (11/13) and AML (21/24) than in ALL (12/20 patients). ALL patients also showed significantly lower methylation densities of HOXC4 (median 12%, range 5–39%) than AML patients (median 32%, range 4–54%, p=0.001), underscoring the importance of HOXC4 for lymphoid cells. Unsupervised hierarchical clustering of methylation densities of all analyzed HOX genes clearly separated AML from ALL patients. We propose that epigenetic silencing of HOX genes by DNA methylation plays a role in the process of leukemic transformation of hematopoietic cells. Further analyses will help to gain a better insight into the role of HOX gene dysregulation in leukemia.

Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 492-492
Author(s):  
Wei-Gang Tong ◽  
William G. Wierda ◽  
Neby Bekele ◽  
Shao-Qing Kuang ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cpg Islands ◽  
Human Leukemia ◽  
Dna Hypomethylation ◽  
Line P ◽  
Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

Digital Restriction Enzyme Analysis of Methylation (DREAM) by Next Generation Sequencing Uncovers Epigenetic Disturbances In Acute Myelogenous Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3635-3635
Author(s):  
Frank Neumann ◽  
Jean-Pierre Issa ◽  
Yue Lu ◽  
Marcos R Estecio ◽  
Rong He ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cpg Islands ◽  
Leukemia Cell ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Cpg Sites ◽  
Leukemia Cell Lines ◽  
Normal Controls

Abstract Abstract 3635 DNA methylation is a key epigenetic mark affecting the configuration of chromatin and the potential for gene expression. Disorganization of DNA methylation contributes to the development of leukemia. There is a need for high resolution, quantitative and cost effective methods to investigate changes of methylome in leukemia. To achieve this goal, we have recently developed a digital restriction enzyme analysis of methylation (DREAM) for quantitative mapping of DNA methylation at approximately 50,000 CpG sites across the whole genome (Jelinek et al., ASH 2009, abstract 567). The method is based on creating distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of genomic DNA with the SmaI and XmaI endonucleases and on resolving these signatures by massively parallel sequencing. Using the DREAM method, we have analyzed DNA methylation in bone marrow cells from 2 patients with AML, 3 samples of white blood cells from healthy adults and 2 myeloid leukemia cell lines (K562 and HEL). The first patient (Pt#1) was a 72 year-old male with AML transformation of the myelodysplastic syndrome (MDS). He had 32% blasts in the bone marrow and a complex karyotype. He had received lenalidomide treatment only. The second AML patient (Pt#2) was a 28 year-old male suffering from a relapse of an AML FAB M1. The bone marrow showed 87% of blasts and a complex karyotype. The patient was heavily pretreated with daunorubicin, ara-C, etoposide, 6-thioguanine, dexamethasone and l-asparaginase. Neither of the patients received demethylating drugs. Using typically 2 sequencing lanes per sample and paired-end reads of 36 bases on the Illumina Gene Analyzer II platform, we acquired 20–38 (median 33) million sequence tags per sample; of these, 7–17 (median 12) million were mapped to SmaI/XmaI sites unique in the human genome. With a threshold of minimum 20-fold coverage, we obtained quantitative information on the DNA methylation level of 39,603-53,312 (median 44,490) CpG sites associated with 8,939-10,735 (median 9,517) genes. In general, methylation was largely absent within CpG islands (CGI). The CpG sites most protected from methylation were in CGI and within 1 kb from gene transcription start sites (TSS). These regions were represented by 13,474 CpG sites. Focusing our analysis on these CpG sites, methylation >10% was detected only in 268 sites in normal controls (1.9%). The numbers of sites with methylation >10% were significantly higher (P<.0001, chi-square test) in both AML patients: 397 sites in Pt#1 (2.9%) and 2,143 sites in Pt#2 (15.6%), respectively. Leukemia cell lines mirrored the pattern of CGI hypermethylation seen in primary AML cells. Methylation >10% in CGI within 1 kb from TSS was observed at 2,331 sites (17.0%) in K562 and at 2,484 sites (18.1%) in HEL. Differential hypermethylation in AML patients affected 906 genes, including multiple genes previously shown to be methylated in cancer, such as CDKN1B, FOXO3, GATA2, GATA4, GDNF, HOXA9, IGFBP3, SALL1 and WT1. Methylated genes were significantly enriched in canonical pathways affecting embryonic stem cell signaling, Wnt-beta-catenin signaling and pluripotency suggesting an important role in AML stem cells. In contrast to CGI, it is known that CpG sites outside of CpG islands (NCGI) are generally fully methylated in normal cells. We analyzed 11,220 NCGI sites that were >1 kb from gene TSS. Methylation >90% was observed at 5,217 (46%) sites in normal controls, in 5,380 sites (48%) in Pt#1, while only in 1,873 sites (17%) in Pt#2 (P<.0001). Leukemia cell lines also showed this NCGI hypomethylation with only 1,422 (13%) fully methylated sites in K562 and 4,200 sites (37%) in HEL. Thus, significant degrees of hypomethylation in NCGI were observed in Pt#2, and in K562 and HEL cell lines, but not in Pt#1. In conclusion, high resolution quantitative mapping of DNA methylation changes in leukemia is feasible using the DREAM method. Relatively small alterations in DNA methylation observed in the MDS/AML Pt#1 contrasted with extensive hyper and hypomethylation found in Pt#2 with relapsed AML M1. Our results illustrate the complexity and diverse extent of DNA methylation changes in leukemia. Disclosures: No relevant conflicts of interest to declare.

Complex Regulation of the Homeobox Transcription Factor TGIF: Differential Expression of Multiple 5′ Isoforms.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4254-4254
Author(s):  
Rizwan Hamid ◽  
Johnequia Patterson ◽  
Danko Martincic ◽  
Stephen J. Brandt
Keyword(s):  
Cell Lines ◽  
Expressed Sequence Tag ◽  
Genomic Structure ◽  
Complex Structure ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Splice Isoforms ◽  
Multiple Promoters ◽  
Leukemic Cell Lines

Abstract TG-interacting factor (TGIF) is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins. In addition to its involvement by mutation or deletion in the inherited craniofacial disorder holoprosencephaly, we have shown that expression of TGIF is highly predictive of relapse and survival in acute myelogenous leukemia (AML). To better understand how TGIF expression is regulated, we characterized its genomic structure using expressed sequence tag analysis, reverse transcriptase-coupled PCR, and rapid amplification of cDNA ends. These studies revealed a complex pattern of gene expression, with 15 splice isoforms and 11 alternative 5′ exons spread over 40 kb of DNA, suggesting the presence of multiple promoters. Real-time and semi-quantitative PCR analysis showed these isoforms were differentially expressed in various adult tissues, leukemic cell lines, and AML blasts. Further, isoform C was found to be the major RNA product in hematopoietic cells, contributing significantly to total TGIF expression in leukemic cell lines TF1a, U937, AML-193, KG-1a, Kasumi-1, K562, GDM-1, HL-60 and AML blasts. This analysis suggests that altered splicing and/or expression of specific isoforms could be responsible for the reduced levels of TGIF message observed in AML blasts and cell lines. The unusually complex structure of the TGIF gene may enable its precise regulation during normal hematopoiesis and may be relevant to its reduced expression in myeloid leukemias.

Aberrant CpG Island Methylation in AML Is Associated with Disease Progression and Relapse.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 564-564
Author(s):  
Heike Kroeger ◽  
Jaroslav Jelinek ◽  
Carlos E. Bueso-Ramos ◽  
Jean-Pierre J. Issa
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Disease Progression ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Cpg Islands ◽  
Myelogenous Leukemia ◽  
Peripheral Blood Cells ◽  
Z Score ◽  
Methylation Index

The role of DNA methylation in relapse and progression of acute myelogenous leukemia (AML) is incompletely understood. We studied DNA methylation of 9 promoter-associated CpG islands of genes frequently hypermethylated in leukemic cell lines. These were NOR1, NPM2, HIN1, SLC26A4, CDH13, PGRA, PGRB, OLIG2 and the tumor suppressor gene p15INK4b. We examined bone marrow and/or peripheral blood cells collected at the time of diagnosis and at the first relapse from 32 patients (13 females, 19 males) with AML. The median age was 58 years (20–68), the median survival was 18 months (8–80), the median blast count was 64% (20–98), and 10 patients had additional solid tumors and/or lymphatic/hematologic malignancies. Bisulfite treatment of DNA, followed by PCR and pyrosequencing were used to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands. We analyzed methylation data for individual genes and for a methylation index derived after Z-score (z = [value –mean]/standard deviation) transformation to equalize absolute differences between individual genes and we used paired t-tests for statistical analysis. Abnormal hypermethylation (≥10%) in bone marrow and peripheral blood cells at diagnosis was detected in all 9 investigated genes, with a range of 7/29 (24%) for HIN1 to 24/32 (75%) for CDH13. On average, an increase in methylation between diagnosis and relapse was detected in all genes, and was significant for CDH13 (mean 10%, p=0.0006), SLC26A4 (mean 7%, p=0.0012), HIN1 (mean 8%, p=0.0037), NPM2 (mean 7%, p=0.0073), p15INK4b (mean 13%, p=0.0081), NOR1 (mean 4%, p=0.0124), PGRB (mean 6%, p=0.0144), PGRA (mean 9%, p=0.0275), and OLIG2 (mean 3%, p=0.0732). When analyzed by change in methylation status: negative (methylation below 10%) turning positive (methylation ≥ 10%) and vice versa, of 238 analyses, 39 (16%) showed a negative to positive switch, 15 (6%) showed a positive to negative switch, and the remaining 184 (77%) were either positive or negative unchanged. Finally, when analyzed in individual patients, an increase in methylation was noted in 29 of 32 patients (91%). The median increase in methylation index between diagnosis and relapse calculated as a delta-Z-score was 30% (range from −10% to 147%), and was highly significant (p<0.0001). In summary, abnormal hypermethylation in bone marrow and/or peripheral blood cells from AML patients was detected in all investigated genes at diagnosis. Methylation levels further increased at relapse of the disease in 29 of 32 patients in 1 to 8 of 9 investigated genes. Based on quantitative analyses, we propose that methylation of CDH13, PGRB, PGRA and OLIG2 CpG islands are early markers for AML, while hypermethylation of HIN1, NPM2 and p15INK4b CpG islands is associated with disease progression and predominantly appears at relapse. Thus, aberrant hypermethylation is clearly associated with disease progression and relapse in AML, and likely mediates drug resistance in this setting. Increase of Methylation Index in Relapse Increase of Methylation Index in Relapse

scholarly journals Antigenic analysis of hematopoiesis. I. Expression of the My-1 granulocyte surface antigen on human marrow cells and leukemic cell lines

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1222-1231 ◽  
Author(s):  
LC Strauss ◽  
RK Stuart ◽  
CI Civin
Keyword(s):  
Cell Lines ◽  
Surface Antigen ◽  
Blood Cells ◽  
Marrow Cell ◽  
Large Fraction ◽  
Leukemic Cell ◽  
Antigenic Analysis ◽  
Leukemic Cell Lines ◽  
Normal Human ◽  
Myeloid Leukemic Cell

Abstract Five monoclonal antibodies that identify the My-1 human granulocyte surface antigen were not reactive with other peripheral blood cells. These antibodies effected complement-dependent cytolysis of a large fraction of normal human marrow leukocytes. This My-1-positive marrow cell population consisted of morphologically identifiable granulocytic precursor cells. Colony-forming cells of the granulocyte-monocyte lineage (CFC-GM) did not express My-1, suggesting that the My-1 antigen is expressed later in normal granulocytic maturation. However, these antibodies did react with myeloid leukemic cell lines. The significance and potential utility of these probes for the understanding of granulopoietic differentiation is discussed.

scholarly journals Lineage infidelity of a human myelogenous leukemia cell line

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1059-1063 ◽  
Author(s):  
A Palumbo ◽  
J Minowada ◽  
J Erikson ◽  
CM Croce ◽  
G Rovera
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Light Chain Gene ◽  
Leukemic Cell Lines ◽  
Atypical Cells

Abstract We have analyzed the organization and expression of the immunoglobulin heavy and light chain gene in the human myeloblastic leukemic sublines, ML1, ML2, and ML3, and in the human myeloid leukemic cell lines, HL-60, U937, THP1, and K562. ML1, ML2, and ML3 cells, despite a predominant granulocytic phenotype, express a rearrangement of the immunoglobulin heavy chain gene that typically occurs during the early stages of the B cell differentiation pathway. No rearrangement was found in any of the other cell lines tested. These findings strongly support the notion that, at least in some cases, acute myeloid leukemia (AML) cells represent highly atypical cells with profoundly altered gene expression, rather than cells arrested at a well-defined stage of the myeloid lineage.

scholarly journals Antigenic analysis of hematopoiesis. I. Expression of the My-1 granulocyte surface antigen on human marrow cells and leukemic cell lines

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1222-1231
Author(s):  
LC Strauss ◽  
RK Stuart ◽  
CI Civin
Keyword(s):  
Cell Lines ◽  
Surface Antigen ◽  
Blood Cells ◽  
Marrow Cell ◽  
Large Fraction ◽  
Leukemic Cell ◽  
Antigenic Analysis ◽  
Leukemic Cell Lines ◽  
Normal Human ◽  
Myeloid Leukemic Cell

Five monoclonal antibodies that identify the My-1 human granulocyte surface antigen were not reactive with other peripheral blood cells. These antibodies effected complement-dependent cytolysis of a large fraction of normal human marrow leukocytes. This My-1-positive marrow cell population consisted of morphologically identifiable granulocytic precursor cells. Colony-forming cells of the granulocyte-monocyte lineage (CFC-GM) did not express My-1, suggesting that the My-1 antigen is expressed later in normal granulocytic maturation. However, these antibodies did react with myeloid leukemic cell lines. The significance and potential utility of these probes for the understanding of granulopoietic differentiation is discussed.

Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4417-4417
Author(s):  
Marius Stiefelhagen ◽  
Marlon R. Veldwijk ◽  
Anna Jauch ◽  
Volker Eckstein ◽  
Stephanie Laufs ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Transfer Rates ◽  
Primary Material ◽  
Leukemic Cell Lines

Abstract Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells. Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH. Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH. Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.

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