Cell lines and culture
The human cell lines K562 (chronic myeloid leukemia; ACC-10), Jurkat (T-ALL; ACC-282), Molt-4 (T-ALL; ACC-362) and KG-1a (erythroleukemia; ACC-421) were purchased from the DSMZ (Braunschweig, Germany). Cells were maintained in RPMI1640 medium containing 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Merck Millipore, Darmstadt, Germany). Medium was supplemented with 10 % fetal bovine serum (20 % for Molt-4 cells; Linaris, Bettingen, Germany). Cell cultures were routinely checked for mycoplasma contamination using PCR (Merck Millipore).
Plasmid constructs and transfection
Human cDNA prepared from normal BMSCs served as template for a standard PCR (Life Technologies, Regensburg, Germany) to amplify the IGFBP7 transcript variant1 (accession number: NCBI NM_001553.2) using the primers 5’-CACCCCGCCATGGAG-3’ and 5’-TATAGCTCGGCACCTTCACC-3’. The 857 bp PCR product was cloned into vector pcDNA3.1myc/HisC (Life Technologies, Regensburg, Germany) for eukaryotic over-expression (pIGFBP7). Jurkat and Molt-4 cells were transfected with pIGFBP7 or the empty vector for as control (pCntrl) using the Nucleofector device (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. After 24 hours, transfected cells received neomycin (800 μg/mL; Merck Millipore, Darmstadt, Germany) for three weeks before IGFBP7 over-expression was confirmed by qRT-PCR. Cells were cloned by limiting dilution into 96-well plates (Nunc, Glostrup, Denmark). Cultures were kept in the presence of 800 μg/mL neomycin for an additional 4 weeks. Before conductions of experiments, cells were thawed, cultured for two weeks in the presence of neomycin and stable IGFBP7 over-expression reconfirmed by qRT-PCR.
RNA extraction and quantitative real-time PCR
RNA was isolated with the RNeasy Kit (Qiagen, Hilden, Germany) and transcribed into cDNA using MMLV reverse transcriptase (Epicentre, Chicago, USA). Quantitative real-time PCR (qRT-PCR) for IGFBP7 was performed as previously described in a Sybr Green (Invitrogen GmbH, Karlsruhe, Germany) PCR assay  using the primers IGFBP7-forward: 5’-CATCACCCAGGTCAGCAAG-3’ and IGFBP7-reverse: 5’-TCACAGCTCAAGTACACCTG-3’. To measure IGF1R mRNA expression primers IGF1R-forward 5’-ACGGGGCGATCTCAAAAGTT-3’ and IGF1R-reverse 5’-CTCTCCGGCCATCTGAATCA-3’ were used. Expression of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. Relative Expression values were expressed as 2-ΔCT using the comparative cycle threshold (ct) method .
IGFBP7 as secreted protein is likely to act through paracrine/autocrine pathways; therefore its concentration in the conditioned medium from IGFBP7 over-expressing versus control cell lines was measured with a “Super-X” IGFBP7-ELISA kit (Antigenix America Inc., NY, USA) along the manufacturer’s instructions. To harvest conditioned medium, transfected cells were seeded at 1×106 cells/ml and grown for four days. The cells were then separated from the medium by centrifugation for 10 min at 2000 rpm at 4 °C and the medium was treated with cOmplete Mini protease inhibitor cocktail (Roche, Basel, Switzerland) and stored at −80 °C until analysis.
WST-1 proliferation assays
To determine the effect of IGFBP7 over-expression on proliferation of leukemic cell lines, the pCntrl or pIGFBP7 transfected clones were seeded in triplicates in different 96-well plates for separate time points. 1×105 cells/well in 100 μl were seeded and incubated at 37 °C, 5 % CO2 for one to four days. At the endpoint of the experiment, 10 μl WST-1 reagent (Roche, Basel, Switzerland), diluted 1:2 with regular medium, was added to each well. Plates were then incubated for two hours to allow for the reduction of WST-1 reagent to soluble formazan by the respiratory chain of the viable cells. Absorbance was measured with a Sunrise (Tecan, Männedorf, Switzerland) microplate absorbance reader at 450 nm with a reference wavelength of 620 nm.
Apoptosis of cells treated with drugs or starvation-induced apoptosis was measured by staining for AnnexinV-phycoerythrin (FITC) and 7-amino-actinomycin D (7-AAD) using an AnnexinV–FITC apoptosis detection kit (BD Pharmingen, Heidelberg, Germany). For chemosensitivity-assays cells were seeded at 1×106 cells/ml in triplicates and treated with vincristine (1 ng/ml), etoposide (1 μg/ml), cytarabine (araC) (1 μg/mL) or asparginase (1 IU/ml) in combination with DMSO alone or 500 nM IGF1-R-inhibitor NVP-AEW541 (AEW451; Novartis AG, Basel, Switzerland) dissolved in DMSO. Drug concentrations used in the assays were determined in prior WST-1 assays of different concentrations. Cells were harvested after 24 h and analyzed by FACSCalibur (BD Pharmingen, Heidelberg, Germany).
BrDU cell cycle assays
Cell cycle analysis was performed using a BrDU-FITC kit (BD Pharmingen, Heidelberg, Germany) as described in manufacturer’s protocol. Before staining and FACS analysis, cells were incubated with 10 μM BrDU for four hours.
IGF1-R FACS staining
IGF1-R surface expression was measured by a flowcytometric assay. Cells were seeded at 1×106 cells/ml in duplicates, cultured normally for four days and stained with anti-IGF-IR antibody, clone αIR3 (Merck Millipore, Darmstadt, Germany) which recognizes the ~130 kDa α and the ~90 kDa β subunits of the IGF1-R. For isotype controls an unspecific mouse IgG1 was used (Santa Cruz Biotechnology, Dallas, USA). For FACS analysis samples were stained with anti-mouse IgG Alexa®488 (New England Biolabs, Ipswich, USA) as secondary antibody. The median fluorescence intensity (MFI) of the samples was normalized to the MFI of the isotype controls before pooling values of independent experiments.
Cells were seeded at 1×106 cells/ml, incubated for two days, and lysed in RIPA buffer [20 mM Tris–HCl, 150 mM NaCL, 1 mM NaCl, 1 mM Na2-EDTA, 1 mM EGTA, 1 % NP-40, 1 % sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4 and cOmplete Mini protease inhibitor cocktail (Roche, Basel, Switzerland)]. Whole cell extracts of 5×105 cells were diluted in SDS-loading buffer and denatured for 5 min at 95 °C. The samples were then separated by 12.5 % SDS-polyacrylamide gel electrophoresis and blotted onto a 0.2 μm PDV membrane (Bio-Rad Laboratories Inc., Hercules, USA). After blocking in TBST 3 % BSA, blots were incubated with IGF-IRβ antibody (C-20, Santa Cruz Biotechnology, Dallas, USA) following by anti-rabbit-HRP (Santa Cruz Biotechnology, Dallas, USA) and developed with an ECL system (Western Lightning Plus ECL; PerkinElmer, Waltham, USA). As loading control, blots were stripped and incubated again with an anti-beta-actin antibody-HRP (Abcam, Cambridge, UK).
Gene expression profiles
IGF1-R-associated GEP of an independent set of 86 adult T-ALL samples were generated from raw data obtained from the Microarrays Innovations in Leukemia (MILE) multicenter study (HG-U133 Plus 2.0 and HG-U133 A+B; Affymetrix, Santa Clara, CA, USA) . All patients had given written informed consent to participate according to the Declaration of Helsinki and the MILE study design was approved by the ethics committees of the participating institutions . As we have used this already existent dataset and the experiments we performed ourselves only included established cell lines, no additional approval by an ethics committee was necessary. Information about the MILE study is provided in the cited article .
For analysis the samples were divided into a low IGF1-R expression group (n = 42) and a high IGF1-R group (n = 41) according to the expression level of IGF1-R (represented by the median of the two probe sets 203628_at, 203627_at). In order to obtain gene expression signatures that discriminated between high and low IGF1-R expressers, samples in the high IGF1-R expression group were compared to samples in the low expression group. Genes were considered to be differentially expressed if their expression showed at least a 1.5-fold change and a FDR < 0.05. The data analyses were carried out with Partek Genomic Suite 6.6 (Partek Incorporated, St. Louis). For further analysis DAVID Bioinformatics Database was utilized to functionally annotate the genes up- or down-regulated in IGF1R-high patients (Gene Ontology: GOTERM_BP_FAT).
The statistical difference between values of two independent groups was tested using the nonparametric Mann–Whitney U-test. For paired values the non-parametric Wilcoxon matched-pair test was used. A P-value ≤ 0.05 (two-sided) was considered to indicate a significant difference. Statistical analyses were performed with Prism 5 software (GraphPad Software Inc., San Diego, USA).