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Full Text (PDF) Research Article Large-Scale CpG Methylation Analysis Identifies Novel Candidate Genes and Reveals Methylation Hotspots in Acute Lymphoblastic Leukemia Kristen H. Taylor,1 Keila E. Pena-Hernandez,1,2 J. Wade Davis,2,3 Gerald L. Arthur,2 Deiter J. Duff,1 Huidong Shi,1 Farah B. Rahmatpanah,1 Ozy Sjahputera,1 and Charles W. Caldwell1 1Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, 2Department of Health Management and Informatics, and 3Department of Statistics, University of Missouri-Columbia School of Medicine, Columbia, Missouri Abstract tions that modify the function of a gene or set of genes. Many This study examined DNA methylation associated with acute nonrandom chromosomal translocations are known to modify lymphoblastic leukemia (ALL) and showed that selected proliferation, differentiation, apoptosis, and gene transcription in molecular targets can be pharmacologically modulated to ALL (3). reverse gene silencing. A CpGisland (CGI)microarray The aberrant methylation of gene promoter-related CpG islands containing more than 3,400 unique clones that span all (CGI) is an epigenetic modification of DNA that can inappropri- human chromosomes was used for large-scale discovery ately silence or down-regulate gene expression and could have experiments and led to 262 unique CGI loci being statistically deleterious effects if the targeted genes function as tumor identified as methylated in ALL lymphoblasts. The methyla- suppressors. However, the mechanisms by which DNA methylation tion status of 10 clones encompassing 11 genes (DCC, DLC-1, occurs in ALL, when these events occur, and which genes are DDX51, KCNK2, LRP1B, NKX6-1, NOPE, PCDHGA12, RPIB9, involved are not wholly understood. It has been established, ABCB1, and SLC2A14) identified as differentially methylated however, that hematopoietic progenitor cells undergo several between ALL patients and controls was independently verified. molecular changes on interaction with stromal cells, including Consequently, the methylation status of DDX51 was found to the up-regulation of DNA methyltransferase 1 (4), which is involved differentiate patients with B- and T-ALL subtypes (P = 0.011, in both the establishment and the maintenance of DNA meth- Fisher’s exact test). Next, the relationship between methylation ylation (5). Additionally, previous studies, which investigated and expression of these genes was examined in ALL cell lines aberrant methylation in ALL, have associated hypermethylated (NALM-6 and Jurkat) before and after treatments with 5-aza- promoters with prognosis (6), cytogenetic alterations (7), subtype 2-deoxycytidine and trichostatin A. More than a 10-fold (8), and relapse (9). Although the elucidation of the aberrant increase in mRNA expression was observed for two previously methylation profiles involved in ALL has been limited by the small identified tumor suppressor genes (DLC-1 and DCC) and also number of CGIs analyzed to date, these studies suggest that it is for RPIB9 and PCDHGA12. Although the mechanisms that common for multiple promoter CGIs to be aberrantly methylated. lead to the CGI methylation of these genes are unknown, Toward a more global view of the extent to which gene promoter and/or first exon DNA methylation is present in ALL, methylation bisulfite sequencing of the promoter of RPIB9 suggests that microarray profiles were developed using a CGI microarray expression is inhibited by methylation within SP1 and AP2 f transcription factor binding motifs. Finally, specific chromo- consisting of 9K clones (10) representing more than 3,400 unique somal methylation hotspots were found to be associated with sequences spanning all human chromosomes. The patterns of ALL. This study sets the stage for acquiring a better biological aberrant methylation and mRNA expression levels were indepen- understanding of ALL and for the identification of epigenetic dently confirmed in 11 putative candidate genes. Additionally, the biomarkers useful for differential diagnosis, therapeutic chromosomal locations of 262 differentially methylated loci were monitoring, and the detection of leukemic relapse. [Cancer examined to identify the distribution of genomic regions that were Res 2007;67(6):2617–25] methylated in ALL patients. Although this microarray does not include all of the annotated CGI fragments within the human genome, it has proven to be a valuable discovery tool for elucidating Introduction epigenetic mechanisms underlying other cancer types (11, 12) and Acute lymphoblastic leukemia (ALL) arises when B- or T-cell has been used in this study to generate specific ALL epigenetic progenitors fail to differentiate into mature cells and is character- profiles and to provide new insights into ALL cell biology. ized by the rapid proliferation of immature lymphoblasts (1, 2). Several factors may play a role in leukemogenesis, including chromosomal translocations and genetic or epigenetic modifica- Materials and Methods Tissue specimens. Bone marrow samples of patients diagnosed with ALL at the Ellis Fischel Cancer Center (Columbia, MO) were obtained in compliance with the local Institutional Review Board. Leukemic blasts, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). due to their high proportion, were concentrated to >90% purity using Ficoll Requests for reprints: Charles W. Caldwell, Department of Pathology and density gradient centrifugation. DNA was isolated using the QIAamp DNA Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s specifica- Medicine, One Hospital Drive, DC055.07, Columbia, MO 65212. Phone: 573-884-2642; tions from 20 specimens: 6 from patients with precursor T-cell ALL, 10 from Fax: 573-884-4612; E-mail: [email protected]. I2007 American Association for Cancer Research. patients with precursor B-cell ALL, and 4 from healthy donors used as doi:10.1158/0008-5472.CAN-06-3993 controls. www.aacrjournals.org 2617 Cancer Res 2007; 67: (6). March 15, 2007 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research Cell lines. Precursor B-cell ALL cell lines representing various Quantitative real-time MSP. Quantitative real-time MSP (qMSP) was immunophenotypic stages of precursor B-cell development, NALM-6 done with deleted in liver cancer 1 (DLC-1) primers as described previously À (CD10+, CD19+, and CD20 ), MN-60 (CD10+, CD19+, and CD20+), and SD-1 (12). Briefly, 100 ng of bisulfite-treated DNA and ABgene QPCR Mix À (CD10 , CD19+, and CD20+) and the precursor T-cell ALL cell line, Jurkat, (ABgene, Inc., Rochester, NY) were used for PCR amplification as were purchased from the Deutsche Sammlung von Mikroorganismen und recommended by the manufacturer. The reaction was carried out in 40 to Zellkulturen (Braunschweig, Germany) and grown in appropriate medium 45 cycles using a SmartCycler (Cepheid, Kingwood, TX). and resupplemented as necessary with fresh medium. Cells were harvested Cell line treatment. The Jurkat (precursor T cell) and NALM-6 and DNA was extracted using the QIAamp DNA Mini kit. (precursor B cell) ALL cell lines were grown in flasks with RPMI 1640 Preparation of the CGI microarray. The microarray panel containing supplemented with 10% fetal bovine serum, L-glutamine, and gentamicin. 8,640 CGI clones was prepared as described (13). Amplified PCR products Treatment was conducted during the log phase of growth with 5-aza- were spotted in the presence of 20% DMSO, on UltraGap slides (Corning 2-deoxycytidine (5-aza) and/or trichostatin A (TSA), whereas the control Life Science, Acton, MA). The slides were postprocessed immediately before cells were not treated. Jurkat cells were seeded at 8 Â 106/mL and NALM-6 the hybridization using Pronto Universal Microarray Reagents (Corning Life cells were seeded at 5 Â 106/mL. In culture, TSA was added at a 1 Amol/L Science). All CGI clones present on the microarray were sequenced recently final concentration and incubated for 6 h, whereas 5-aza was added at a by the Microarray Centre of University Health Network (Toronto, Ontario, 1 Amol/L concentration and incubated for 54 and 78 h in Jurkat and NALM-6 Canada; ref. 14). (based on different dividing times), respectively, with a medium change Amplicon development and differential methylation hybridization. every 24 h. The cell culture that received both TSA and 5-aza treatment Amplicons were generated and differential methylation hybridization was first incubated with 5-aza as described previously, which was followed (DMH) was done as described previously (10–12). Briefly, 2 Ag of genomic by an additional 6-h incubation with TSA. RNA and DNA from the cultured DNA were digested with MseI followed by ligation of PCR linkers and cells were extracted for use in RT-PCR and COBRA, respectively, using the digestion with methylation sensitive endonucleases (HpaII and BstUI). previously mentioned kits. PCR was then done to amplify only methylated fragments or fragments Quantitative real-time RT-PCR. Total RNA (2 Ag) from treated and containing no internal HpaII or BstUI sites. The amplicons from the ALL untreated cell lines was pretreated with DNase I to remove potential DNA and sex-matched normal control sample, which comprised pooled DNA contaminants and was then reverse transcribed in the presence of from multiple donors (Promega, Madison, WI), were labeled with Cy5 or SuperScript II
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