Leukemia cell lines HL-60, MV4-11, U937, DAMI and K562 were obtained from the American Type Culture Collection (ATCC). CCRF, Raji, Jurkat, 697 and SHI-1 cell lines (gifts from Professor Wang Jian-Rong, The Cyrus Tang Hematology center of Soochow University). The entire cell lines were maintained at 37 °C in the RPMI 1640 (GibcoR, Life Technologies, Carlsbad, CA) supplemented with 10 % fetal bovine serum (Invitrogen, Life Technologies, Carlsbad, CA).
Patients and samples
Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 105 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2006 and 2011. Research involving human subjects, human material, or human data, have been performed in accordance with the Declaration of Helsinki. Ethical approval was provided by the Children's Hospital of Soochow University Ethics Committee (No.SUEC2006-011 and No.SUEC2000-021), and informed consent was obtained from the parents or guardians. AML diagnosis was made in accordance with the revised French–American–British (FAB) classification. Additionally, bone marrow samples from 12 healthy donors and 8 patients with Idiopathic thrombocytopenic purpura (ITP) were analyzed as controls. Bone marrow mononuclear cells (BMNCs) were isolated using Ficoll solution within 2 h after bone marrow samples harvested and immediately subjected for the extraction of total RNA and genomic DNA.
CD34 + cell purification
For CD34+cell selection, the Miltenyi immunoaffinity device (VarioMACS 130-046-703) was used according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA). Briefly, the CD34+ cells are magnetically labeled with CD34 MicroBeads. Then, the cell suspension is loaded onto a MACSR Column which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD34+ cells are retained within the column. The unlabeled cells run through; this cell fraction is thus depleted of CD34+ cells. After removing the column from the magnetic field, the magnetically retained CD34+ cells can be eluted as the positively selected cell fraction.
Analysis of promoter methylation in pediatric AML by NimbleGen Human DNA Methylation arrays
Analysis of the methylation status of genes in five pediatric AML samples (M1, M2, M3, M4 and M5) and three NBM samples (N1, N2, and N3) using NimbleGen Human DNA Methylation arrays. NimbleGen Human DNA Methylation arrays Protocol: Step 1, Genomic DNA Extraction and Fragmentation, Genomic DNA (gDNA) was extracted from 8 samples using a DNeasy Blood & Tissue Kit (Qiagen, Fremont, CA). The purified gDNA was then quantified and quality assessed by nanodrop ND-1000. Step 2, Immunoprecipitation, 1 μg of sonicated genomic DNA was used for immunoprecipitation using a mouse monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heat-denatured at 94 °C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4 °C with rocking agitation in 400 μL immunoprecipitation buffer (0.5 % BSA in PBS). To recover the immunoprecipitated DNA fragments, 200 μL of anti-mouse IgG magnetic beads were added and incubated for an additional 2 h at 4 °C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25 % SDS and 0.25 mg/mL proteinase K for 2 h at 65 °C and then allowed to cool down to room temperature. MeDIP DNA were purified using Qiagen MinElute columns (Qiagen). Step 3, Whole Genome Amplification (WGA). Step 4, DNA Labelling and Array Hybridization, the purified DNA was quantified using a nanodrop ND-1000. For DNA labelling, the NimbleGen Dual-Color DNA Labeling Kit was used according to the manufacturer’s guideline detailed in the NimbleGen MeDIP-chip protocol (Nimblegen Systems, Inc., Madison, WI, USA). Microarrays were hybridized at 42 °C during 16 to 20 h with Cy3/5 labelled DNA in Nimblegen hybridization buffer/ hybridization component A in a hybridization chamber (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA). For array hybridization, Roche NimbleGen's Promoter plus CpG Island array was used, which is a 385 k format array design containing 28,226 CpG Islands and all well-characterized Promoter regions (from about -800 bp to +200 bp of the TSSs) totally covered by ~385,000 probes. This NimbleGen Human DNA Methylation array analysis was performed by KangChen Bio-tech, Shanghai P.R. China.
Sodium bisulphite modification of genomic DNA
High-molecular-weight genomic DNA was extracted from cell lines and biopsies by a conventional phenol/chloroform method. The sodium bisulphite modification procedure was as described with slight modification [26–28]. In brief, 600 ng of genomic DNA was denatured in 3 M NaOH for 15 min at 37 °C, then mixed with 2 volumes of 2 % low-melting-point agarose. Agarose/DNA mixtures were then pipetted into chilled mineral oil to form agarose beads. Aliquots of 200 μl of 5 M bisulphite solution (2.5 M sodium metabisulphite, 100 mM hydroquinone, both Sigma, USA) were added into each tube containing a single bead. The bisulphite reaction was then carried out by incubating the reaction mixture for 4 h at 50 °C in the dark. Treatments were stopped by equilibration against 1 ml of TE buffer, followed by desulphonation in 500 μl of 0.2 M NaOH. Finally, the beads were washed with 1 ml of TE buffer and directly used for PCR.
The methylation status of the GATA4 (NCBI Reference Sequence of GATA4 : NG_008177.2) promoter region was determined by methylation-specific PCR. Primers were designed with Methprimer design tool (http://www.urogene.org/methprimer/). Primers distinguishing unmethylated (U) and methylated (M) alleles were designed to amplify the sequence: GATA4 B M-forward: 5- TTTTTTAATTTTTGTTTGTATATCGT-3; GATA4 B M-reverse: 5- ACTACCTAACACTACCACCCTACGT-3; GATA4 B U-forward: 5- TTTTTTAATTTTTGTTTGTATATTGT-3; GATA4 B U-reverse: 5- CTACCTAACACTACCACCCTACATC-3.
Each PCR reaction contained 20 ng of sodium bisulphite-modified DNA, 250 pmol of each primer, 250 pmol deoxynucleoside triphosphate, 1 × PCR buffer, and one unit of ExTaq HS polymerase (Takara, Tokyo) in a final reaction volume of 20 μl. Cycling conditions were initial denaturation at 95 °C for 3 min, 40 cycles of 94 °C for 30 s, 65 °C (M) or 63 °C (U) for 30 s, and 72 °C for 30 s. For each set of methylation-specific PCR reactions, in vitro-methylated genomic DNA treated with sodium bisulphite served as a positive methylation control. PCR products were separated on 4 % agarose gels, stained with ethidium bromide and visualized under UV illumination. For cases with borderline results, PCR analyses were repeated.
Bisulfite genomic sequencing
Bisulfite genomic sequencing (BGS) was performed as previously described. BGS primers were from +682 to +904 including 17 CpGs. GATA4 F: 5- GGATTGAATGTTTTTTTGGAAGTT-3 and GATA4 R: 5- CCTCCTTTCCTCAACCTAATAACA-3. Amplified BGS products were TA-cloned; and five to six randomly chosen colonies were sequenced. DNA sequences were analyzed with QUMA Analyzer. (http://quma.cdb.riken.jp/).
Leukemia cell cells treated with 5-aza-2'-deoxycytidine
De-methylation was induced with 5-aza-dC (5-Aza, Sigma-Aldrich, St Louis, MO, USA) treatment at a concentration that induced de-methylation of the DNA without killing the cells. Culture media for HL-60 and MV4-11 cells contained 5 μM 5-Aza. DNA and RNA were extracted after 72 h of 5-Aza treatment for the following analysis.
Quantitative reverse-transcription PCR for GATA4
Quantitative real-time PCR was performed to determine the expression levels of GATA4 genes. Total RNA was reverse transcribed using the Reverse Transcription Kit, according to the manufacturer's protocol (Applied Biosystems Inc., Foster City, CA). The real time PCR primers used to quantify GAPDH expression were: F: 5′-AGAAGGCTGGGGCTCATTTG-3′ and R: 5′-AGGGGCCATCCACAGTCTTC-3′ and for GATA4 were: F: 5′- TAGCCCCACAGTTGACACAC-3′ and R: 5′- GTCCTGCACAGCCTGCC −3′. Real-time PCR analysis was according to the MIQE Guidelines and performed in a total volume of 20 μl including 1 μl of cDNA, primers (0.2 mM each) and 10 μl of SYBR Green mix (Roche). Reactions were run on an Lightcycler 480 (Roche) using universal thermal cycling parameters (95 °C for 5 min, 45 cycles of 10 s at 95 °C, 20 s at 60 °C and 15 s at 72 °C; followed by a melting curve: 10 s at 95 °C, 60 s at 60 °C and continued melting). The results were obtained using the sequence detection software of the Lightcycler 480 and analyzed using Microsoft Excel. For quality control purposes, melting curves were acquired for all samples. The comparative Ct method was used to quantify gene expression. The target gene expression level was normalized to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within the same sample (−⊿Ct), the relative expression of GATA4 was calculated with 106 *Log2(-⊿Ct ).
Western blot analysis
Western blot analysis was introduced before . Cellular proteins were extracted in 40 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 1 % (v/v) Triton X-100, supplemented with protease inhibitors. Equal amounts of protein were resolved on 12 % SDS-PAGE gels, and then transferred to a PVDF membrane (Millipore, Bedford, MA). Blots were blocked and then probed with Polyclonal Goat IgG antibodies against GATA4 (1:1000, R&D. Minneapolis, MN) and GAPDH (1:5000, Sigma, St. Louis, MO). After three times’ washing, blots were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies and visualized by enhanced chemiluminescence kit (Pierce, Rockford, IL). Protein bands were visualized after exposure of the membrane to Kodak X-ray film.
SPSS v11.5 (SPSS Inc., Chicago, IL) was used for statistical analysis. Data are presented as means ± standard deviation. Group t-test was used to compare the expression of GATA4 between DMSO group and 5-Aza group. Statistical significance between methylated sample data and clinical pathological features of AML patients were analyzed by Pearson chi-square test or Fisher's exact test. Statistical significance of GATA4 expression among NBM and pediatric AML groups was determined using one-way ANOVA. A p <0.05 was considered statistically significant.