Chemicals and antibodies
Antibodies to the ectodomain of L1CAM (monoclonal antibody (mAb) L1-11A, a subclone of UJ127.11) and L1-9.3 were described before [
15,
16]. Antibodies for detection in Western blot were as follows: GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany), Acetyl-H3 (9765, New England Biolabs), MAGE-A4 (WH4103M1, Sigma-Aldrich, Taufkirchen, Germany), MAGE-A3 (NBP1-02506, Novus Biologicals, Littleton, USA) and Ny-ESO-1 (Invitrogen, Eggenstein, Germany). 5-AzaC, TSA and VA were obtained for Sigma-Aldrich and dissolved in serum-free medium or DMSO.
RNA extraction from cell lines and Reverse transcriptase reaction were described before [
14]. Specific primers and probes for L1CAM, MAGE-A4, NY-ESO-1 and β-actin as internal standard were determined with the computer program “Primer Express” (Applied Biosystems, Foster City, CA). To prevent amplification of contaminating genomic DNA, the probe was placed at a junction between two exons. Primers were produced by Sigma-Aldrich. All primers were used in a concentration of 300 μM. The sequences for the primers were as follows:
L1CAM forward 5′-ACGAGGGATGGTGTCCACTTCAAA-3′, L1CAM reverse 5′-TTATTGCTGGCAAAGCAGCGGTAG-3′; β-actin forward 5′-ACAAGATGAGATTGGCATGGC-3′, β-actin reverse 5′-GCCACATTGTGAACTTTGGGG-3′; 5′-DNMT1 forward AAGAACGGCATCCTGTACGGAGTT-3′, DNMT1 reverse 5′-TGCTG CCTTTGATGTAGTCGGAGT-3′; MAGE-A3 forward 5′-AGCAAAGCTTCCAGTTCCTTGCAG-3′, MAGE-A3 reverse 5′-ACAGTCGCCCTCTCTTGCGATTAT-3′; MAGE-A4 forward 5′-TAATCCTGCGCGCTATGAGTTCCT-3′, MAGE-A4 reverse 5′-TGACCACATGCTCCAGGACTTTCA-3′; NY-ESO-1 forward 5′-AGTTCTACCTCGCCATGCCTTT-3′, NY-ESO-1 reverse 5′-TCGGATAGTCAGTATGTTGCCGGA-3′.
To determine the mRNA expression levels, 10 ng of cDNA was analysed in triplicates. The PCR reactions were performed with the SYBRgreen Master Mix from Applied Biosystems using an ABI 7300 analyser.
siRNA transfection
24 h before siRNA treatment 1.5 × 105 cells were seeded per 6-well. The transfection was carried out with Interferin (Polyplus, Illkirch, France) following the manufacturer’s protocol. For each well the final siRNA concentration was 10 nM. After the first transfection the cells were incubated for 72 h under standard conditions and then transfected again and analyzed 48 h after the second transfection. siRNA’s used for the knock-down were as follows: siDNMT1 5′-AGACCAGGAUGAGAAGAGA-3′, siGFP 5′-GGCCAGGUCCAGCAGCGCACC-3′. All siRNA’s were synthesized by MWG Eurofines (Ebersberg, Germany).
Treatment of cells and biochemical analysis
Cells were seeded in 6-well plates and treated for 5 days with 5-AzaC or for 24 h with TSA or VA, respectively. After treatment, the cells were lysed for 15 min at 4°C in RIPA lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml leupeptin, 5 μg/ml aprotinin, 1% NP40, 0.5% deoxycholic acid sodium salt, 0.1% SDS) and sonified. After centrifugation at 10000 × g for 10 min at 4°C, supernatant was collected and protein concentrations were determined with a commercial protein assay (Pierce, BCA protein assay, Thermo Scientific, Waltham, USA). For Western blot analysis, 50 μg of protein per lane was separated on 10 or 12% SDS-polyacrylamide gels under reducing conditions and transferred onto Immobilon membranes (Millipore, Germany). Protein loading was controlled by Ponceau red staining of the membranes. After blocking for one hour in Tris-buffered saline (TBS) supplemented with 5% non-fat milk and 0.1% Tween 20 (Sigma-Aldrich GmbH, Taufkirchen, Germany), membranes were incubated for one hour at room temperature in blocking buffer containing the respective primary antibody. Membranes were washed three times in TBS-Tween and incubated for one hour with horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody. Immunodetection was performed with a chemoluminescence system (ECL, GE Healthcare, Freiburg, Germany). Protein band intensities were defined as the mean of pixels within the area (mean) of the band limited by a preformed rectangular area (area) after subtraction of the background pixels. Quantification was carried out using the “ScionImage” (Scion Corp.) software.
Patient cohort and immunohistochemistry
Normal testicular tissue of 10 patients (age range 23–75, median 32) who were orchidectomied between 1994–1996 at the University Hospital Zurich was assembled on a tissue microarray. All patients were resected because of primary testicular germ cell tumors or primary funicular or paratesticular neoplasia (myxoid liposarcoma, well differentiated liposarcoma, monophasic synovial sarcoma). The project has been approved by the local ethics committee (Kanton of Zurich reference number StV 25–2008).
Mouse mAb to MAGE-A4 was kindly provided by the Ludwig Institute for Cancer Research and diluted 1:50. Mouse mAb to NY-ESO1 (Zymed Laboratories Inc.) was diluted 1:50. Mab to L1CAM (clone 14.10) was diluted 1:200. Two protocols were applied: First, on a Ventana Benchmark® platform (Ventana Medical Systems, Tucson, AZ, USA). Here the pretreatment with 60 min boiling in pH 8 Tris buffer was followed by incubation with primary mAb (MAGEA4, NY-ESO1) for 60 min at room temperature (RT) and development with the Ultraview-HRP kit, including incubation with respective secondary antibody for 16 min at RT. Second, on a Leica Bond® platform (Vision Biosystems, Melbourne, Australia), the H2 standard pre-treatment with 60 min boiling in pH8 Tris buffer was followed by incubation with primary mAb (L1CAM for 30 min at RT and development with the Refine-DAB Bond kit, including incubation with secondary antibody for 30 min at RT and additional polymer amplification. All primary antibodies were diluted in Tris/BSA and all staining conditions were predetermined. For negative control the primary antibody was omitted. For both systems, hematoxylin counterstains were applied. H&E staining were performed according to standard protocols.
Microdissection, DNA isolation and methylation analyses
ECs were collected at the Department of Gynecology and Obstetrics, Medical University of Innsbruck. The project has been approved by the local ethics committee (University of Innsbruck ,UN3801; reference number: 282/4.12).
In total, we analyzed 9 endometrioid ECs (8 endometrioid ECs with areas of squamous differentiation), 7 clear cell ECs, 10 papillary serous ECs and 4 normal endometrial tissues. Immunohistochemistry for L1CAM was conducted as described above. DNA from punch biopsies was isolated using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). Not from all tissue samples DNA of high enough quality for further analysis could be recovered. Therefore we restricted our analysis to those tumors where paired samples from L1CAM positive and L1CAM negative areas were available. Genomic DNA from cell lines was isolated using the AllPrep DNA/RNA/protein kit from Qiagen (Hilden, Germany). Bisulfite modification was performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. MethyLight analysis was done as described previously [
17]. Briefly two sets of primers and probes, designed specifically for bisulfite-converted DNA, have been used: a methylated set for the gene of interest and a reference set, collagen, type II, alpha 1 (
COL2A1), to normalize for input DNA. Specificity of the reactions for methylated DNA has been confirmed separately using SssI (New England Biolabs)-treated human white blood cell DNA (heavily methylated). The percentage of fully methylated molecules at a specific locus was calculated by dividing the
GENE:
COL2A1 ratio of a sample by the
GENE:
COL2A1 ratio of SssI-treated controls and multiplying by 100. Primers and probes for
COL2A1 have been described before [
18]. Primers and probes for L1CAM were determined with the assistance of the computer program Primer Express version 2.0.0 (Applied Biosystems, Foster City, CA, USA) to produce a 68-base-pair PCR amplicon (nucleotide positions c4070008-4069940 as defined by GenBank accession number NT_167198.1; -10,671 nucleotides to −10,603 nucleotides upstream from the transcription start site). Genomic DNA not treated with bisulfite (unmodified) was not amplified with the primers (data not shown). Primer sequences were: L1CAM forward 5
′-AATACTCCCTTAACCTCGACCTAACC-3
′, L1CAM reverse 5
′-GGCGTTGCGTGTAGGTGTT-3
′; L1CAM Taq Man probe 5
′FAM-TCGACGACGCCGACCAACGAT-3
′BHQ1. The amplicon was placed in the promoter 1 region [
14].
For L1CAM-bisulfite sequencing the following primers were used:
PP1 forward 5′-TTAGAGAGTTGGAGGAAAATTTG-3′, PP1 reverse 5′-ACACACACACACAAAACAAAAC-3′; PP2 forward 5′-GAGTTTTGTTTTGTGTGTGTGTG-3′, PP2 reverse 5′-CACCCTAACCCCTAATACCAAC-3′, PP3 Forward 5′-AGTAGTTGAAGGGAGTTTGG-3′, PP3 reverse 5′-TAAAAAAAACCCAAAACCTC-3′. The primers were determined with the assistance of the computer program Methyl Primer Express software v1.0 (Applied Biosystems, Foster City, CA, USA).
PCR reactions were performed in a final volume of 50 μl containing 2 U of HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany), 0.2 μM dNTP mix (Qiagen), 250nM of each primer, 1x buffer and 150 ng of bisulfite modified DNA. The thermal cycling conditions comprised an initial denaturation step at 95°C for 15 min, 35 cycles at 94°C for 1 min, 55°C, 58°C or 54°C respectively (for PP1, PP2 or PP3 respectively) for 45 sec and at 72°C for 1 min, and after the last cycle an incubation step at 72°C for 10 min. For visualization and statistical analysis of the raw bisulfite sequencing data the free BiQ Analyzer tool was used [
20].