Human glioma cell lines LN18 and LN229 were grown in Dulbecco's modified eagle medium (DMEM) (Gibco-Life Technologies, Eggenstein, Germany) supplemented with 5% fetal calf serum (FCS) (Seromed, Berlin, Germany), 1% penicillin/streptomycin (Serva, Heidelberg, Germany) and 1% L-glutamine (Gibco-Life Technologies, Eggenstein, Germany).
The entire human MIF cDNA was cloned in antisense orientation into the BamH1/EcoRV restriction sites of the pcDNA 3.1/Myc-His vector (Gibco-Life Technologies, Eggenstein, Germany).
The LN18 cells were transfected at semiconfluent cell density with the linearized antisense and control plasmid with the Lipofectamin reagent (Gibco-Life Technologies, Eggenstein, Germany). Stable transfectants were selected by adding 1 mg/ml G418 (Invitrogen, Leek, Netherlands) to the cultures. After 4 weeks, the remaining cells were plated out highly diluted and the emerging clones picked with a sterilized needle and further propagated. From the initially picked 24 antisense clones (Additional file 1), two of these (termed as1 and as2) were chosen for the experiments because of their high consistent MIF antisense production. Seven empty vector transfected clones were generated in a similar way and clone 5 (here after termed c1) was used as a control for all experiments with the antisenseMIF clones.
RNA preparation and Northern blot analysis
Total RNA was prepared by the TRIzol method (Gibco-Life Technologies, Eggenstein, Germany). RNA samples (5 μg) were separated on 1% agarose gels. The RNA was blotted with 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) onto a positively charged nylon membrane (Boehringer GmbH, Mannheim, Germany). After UV-cross-linking, hybridization was performed under continuous rotation in a hybridization oven (Biometra, Goettingen, Germany). The membranes were hybridized with digoxigenin (DIG)-labeled antisense riboprobes overnight under highly stringent conditions in 50% formamide at 68°C and finally washed in 0.1% SSC, 0.1% sodium dodecyl sulfate at the same temperature. Hybridized DIG-labeled riboprobes were visualized with the DIG nucleic acid detection kit (Boehringer) and CPD-Star chemiluminescence substrate (Tropix, Bedford, Mass.; distributed by Serva, Heidelberg, Germany). Equal loading of the RNA was confirmed by staining of 18S and 28S RNA wth methylene blue. Quantification of band intensities were done with ImageJ (NIH, Bethesda, USA) and normalized to 18S RNA. Results represent the mean of at least 4 independent experiments.
Protein samples were prepared by lyszing cells in 1% Triton containing proteinase and phosphatase inhibitors. Supernatans were concentrated 20-fold with 10 kDa Centricon filter columns (Millipore, Billerica, USA) prior to addition of sample buffer. Western blotting was performed by the NuPAGE electrophoresis system (Novex, San Diego, USA) using 4 to 12% N, N -methylenebisacrylamide-Tris gels. Proteins were transferred onto Optitran BA-S83 membranes (Schleicher & Schuell, Dassel, Germany). The antibodies used were polyclonal anti-human MIF (1:4000) rabbit immunoglobulin G (IgG) as described earlier , anti-p21 (1:1000), anti-p27 (1:1000), anti-C/EBPalpha (1:1000), anti-CD74 (1:500), anti-Erk2 (1:1000), anti-phospho-Erk (1:2000), anti-pan-Akt (1:1000), anti-beta-Actin (1:2000) (all from Santa Cruz, La Jolla, USA), anti-p53 (1:1000), anti-phospho-Akt (1:2000) (all from Cell Signaling, Danvers, USA), anti-CD44 (1:500)(BD Bioscience Pharmingen, Bedford, USA) and peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG diluted 1/2000 in 5% milk in TBS/0,05% Tween (Cell Signalling, Danvers, USA). The bands were visualized by an enhanced chemiluminescence detection system, as recommended by the manufacturer (SuperSignal ULTRA; Pierce, Rockford; USA). Quantification of band intensities were done with ImageJ (NIH, Bethesda, USA) and normalized to Actin if appropriate. Results represent the mean of at least 3 independent experiments.
The cells were plated out in the stated numbers (see figure legend) in 96-well plates (Costar Corporation, Cambridge, USA) and incubated for 24 h prior to the experiments. The cells were incubated for 6 h with fresh medium supplemented with either anti MIF-antibodies or the MIF-inhibitor ISO-1 and then for additional 2 h when the BrdU labeling reagent was added. For experiments with recombinant MIF cells were pre-treated with 10 and 50 ng/ml recombinant human MIF for 12 h before addition of BrdU labeling reagent for another 2 h. The assay was performed using the Cell proliferation ELISA, BrdU chemiluminescence (Boehringer, Mannheim, Germany) according to the manufacturers instructions. The final results were obtained by reading the chemiluminescence values (relative light units (rlu) with a Lumistar automated plate reader (bmg, Offenburg, Germany). Results are given as average of relative values of controls with SD of eight simultaneous experiments. Statistics were calculated using Mann-Whitney unpaired non-parametric test and a p-value < 0.05 was regarded significant. All experiments have at least been repeated three times with similar results.
Over a period of several days, growth was assessed by staining the cells for intracellular protein content. The protein staining was accomplished by using the amido-black method . In brief, the cells were plated out at 5000 cells/well on multiple 96-well plates and fed every other day. For experiments with addition of MIF to culture media, recombinant human MIF was added at 10 ng/ml and 50 ng/ml with every medium change. Every day, the cells in one plate were fixed and denatured with formaldehyde and stained with amido-black solution. After drying of the plate, the amido-black dye was eluted from protein bound with NaOH and the light absorption at 620 nm was recorded with an automated plate reader. Results are given as averages of relative values of controls with SD of eight simultaneous experiments. Statistics were calculated using Mann-Whitney unpaired non-parametric test and a p-value < 0.05 was regarded significant. The experiments have been repeated three times with similar results.
MIF blocking antibody
The antibodies against human MIF were initially raised in rabbits, following a standard immunization protocol. Monoclonal antibodies were then produced from the fusion of myeloma cells and antibody-producing B-lymphocytes (Institute of Biotechnology Vilnius, Lithuania). Binding of the monoclonal antibodies to MIF was confirmed by specific ELISA The antibody was purified from ascites by sepharose columns using Kaptiv-M (Tecnogen, Piana di Monte Verna, Italy).
Small compound MIF inhibitor
The synthetic MIF inhibitor, (S,R)-3-(4-Hydrophenyl)-4,5-dihydro-5-isoazole acetic acid methyl ester (ISO-1) has been recently shown to covalently bind to the D-dopachrome tautomerase activity site of MIF and inhibits several biological activities of MIF in vitro and in vivo . The inhibitor ISO-1 (provided by Y. Al-Abed) was solubilized in DMSO at a concentration of 10 μg/μl (equivalent to 42 mM) and further diluted in PBS if required. For all experiments DMSO only treated cells served as a control.
Recombinant human MIF
Human MIF cDNA was cloned into the pET-17b vector (Novagen, Madison, USA) and expressed in Escherichia coli BL-21 (DE3) strain after induction with 0.4 mM isopropyl -D-thiogalactopyranoside (IPTG) and purified from the soluble fraction of the cell lysate by two-step high-pressure liquid chromatography (HPLC): (i) size exclusion HPLC on Bio-Sil TSK 250 (Bio-Rad, Munich, Germany) and (ii) ion-exchange HPLC on Ultropac TSK CM-3SW (LKB/Pharmacia, Freiburg, Germany). Biological activity has been confirmed using proliferation assay in human fibroblasts (unpublished data) and calcium release assay .
Cells were harvested by trypsinization and blocked in human serum. Staining for CD44 and CD74 was performed using the following antibodies: anti-human CD44-PE (eBioscience, San Diego, USA), anti-human CD74-FITC (BD Bioscience, Bedford, USA) and corresponding isotype controls (eBioscience, San Diego, USA). Analysis was carried out on a BD LSR II (BD, Bedford, USA).