Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 Methods Patients, cell lines and tissue culture The ALL children enrolled in this study were admitted to the Department of Pediatrics, Innsbruck Medical Uni- versity, or the St. Anna Kinderspital in Vienna from September to December 2009, and treated according to the BFM protocol 2000 (for protocol details see: http:// boriskononenko.files.wordpress.com/2009/09/1-proto- koll-all-bfm-2000-mit-amendments-version-26-09-07- d0bad0bed0bfd0b8d18f1.pdf). To analyze the GC response in non-leukemic peripheral blood lymphocytes, Ficoll-purified peripheral blood mononuclear cells from 2 healthy volunteers and 3 children with epilepsia trea- ted with a single injection of GC were included. The study was approved by the Ethics Committees of the Innsbruck Medical University (EK1-1193-172/35 for ALL children and UN2821 for epileptic children) and the Kinderspital in Vienna, and written informed con- sent was obtained from the patients and/or parents or custodians. The characteristics of 3 children with T-ALL and 10 with precursor B-ALL have been published pre- viously [5], the remaining ALL children (3 T-ALLs and 15 precursor B-ALLs) showed similar clinical findings [Rainer et al. in preparation]. All 3 patients with epilep- sia (E3, a 14 year-old girl; E4, a 6 year-old boy; E5, an 11 year-old boy) had an idiopathic generalized epilepsia without any evidence of metabolic, endocrinological, immunological, hematological or infectious disease. The 6 year old girl (epilepsia diagnosed at 4 years of age) was on Valproinate and Clobazam, the 9 year old girl (epilepsia diagnosed at 8 year of age) on Valproinate and Rufinamide, and E5 (epilepsia diagnosed at 2 years of age) on Valproinate and Sultiam. Because of therapy- refractory epilepsia, the 3 patients received their first GC pulse therapy with dexamethasone (Fortecortin, Merck, Vienna, Austria) 20 mg/m2 administered intrave- nously over 1 hour. Blood samples were taken at 2, 6 and 24 hours after drug administration. The T-ALL cell lines CCRF-CEM-C7H2 [18], 6 GC-sensitive and 6 GC-resistant derivatives CCRF- CEM-C7H2 [19], CEM-C7H2-2C8 [20], a CEM-C7H2 derivative with constitutive expression of the tetracy- cline-regulated reverse transactivator, rtTA [21], MOLT4 (CRL-1582, ATCC, Rockville, MD), and Jurkat (untransfected and a rat GR-transfected derivative [22]), the precursor B-cell lines 697/EU-3 (ACC 42, DSMZ, Braunschweig, Germany), NALM6 (ACC 128, DSMZ), RS4;11 (ACC 508, DSMZ) and AT-1 [23], and Burkitt’s lymphoma Daudi (CCL-213, ATCC) were cultured in RPMI 1640 supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37°C, 5% carbon-dioxide and satu- rated humidity. HEK293T packaging cells (ATCC, Man- assas, VA) were cultured in DMEM supplemented as Page 3 of 11 above. The cells were free of mycoplasma infection and their authenticity was verified by DNA fingerprinting, as detailed previously [24]. Doxycycline was dissolved (100 μg/ml) in phosphate-buffered saline and dexa- methasone (10-4M) in 100% ethanol. The final ethanol- concentration in the dexamethasone-treated and control cultures was maintained at 0.1%. All above reagents were from Sigma (Vienna, Austria). Expression profiling Our procedure for generating mRNA expression profiles on Affymetrix HGU133 plus 2.0 microarrays has been detailed previously [5]. Briefly, RNA from peripheral blood lymphoblasts of patients prior to, or at 6-8 or 24 hours after initiation of systemic GC monotherapy (fol- lowing the BFM therapy protocol recommendations), from healthy volunteers or epileptic children 6 hours after GC treatment, and GC-sensitive or GC-resistant cell lines, was converted into labeled target and hybri- dized to Affymetrix HGU133 plus 2.0 microarrays according to standard protocols. The microarray data from the 31 ALL-patients and 5 non-leukemic indivi- duals were normalized separately from those of the 12 cell lines using GCRMA [25] after positive evaluation of comprehensive raw data quality assessment. The analysis was performed in R using packages from Bioconductor [26] version 2.5. Annotation of probe sets to genes based on Affymetrix NetAffx annotation database ver- sion 22 and location of probe sets relative to the 3’ end of the transcripts were manually inspected using the Ensembl genome browser for genes PFKFB1, PFKFB2, PFKFB3 and PFKFB4. Microarray data for the samples of the 13 ALL children and 2 healthy donors were pub- lished previously [5] and are available at NCBI’s GEO database (series GSE2677 and GSE2842). Microarray data for the 12 cell lines (GSE22152) and 4 additional non-leukemic individuals (GSE22779) have been depos- ited at GEO. Apoptosis determinations Apoptosis was determined by FACS analysis of propi- dium iodide (PI)-treated permeabilized cells [27] as pre- viously detailed [28]. Briefly, cells were analyzed with a FACScan cytometer (Becton Dickinson Biosciences, San Jose, CA) in combination with CellQuest Pro software (Becton Dickinson Biosciences) acquiring forward scat- ter/sideward scatter, FL-2 H (log), and FL-3 H (linear). In FL-2 H, the percentage of nuclei with reduced DNA- content (SubG1 peak) was assessed. Immunoblotting Our immunoblotting procedure has been detailed recently [29]. Briefly, proteins were extracted from 5 × 106 cells