FFPE samples, DNA extraction and IDH1 sequencing
The collection of IDH1 mutant tumors consist out of previously published specimen[1, 2, 15] and tumors collected at the neuropathology department of the pathological institute of Heidelberg.
The DNA extractions form FFPE samples were performed by hand using Invisorb Genomic DNA Kit II (Invitek, Berlin, Germany) or semi-automated using Maxwell® 16 FFPE Plus LEV DNA Purification Kit (Promega, Madison, USA) following the manufacturer’s protocol.
The sequencing of published specimen was performed as described in the earlier publications. Our most recent probes were sequenced by generating a fragment of 212 bp length spanning the catalytic domain of IDH1 including codon 100 and 132 using 60 ng each of the sense primer IDH1-JM_f TGATGAGAAGAGGGTTGAGGA and the antisense primer IDH1-JM_r GCAAAATCACATTATTGCCAAC. PCR using standard buffer conditions, 20 ng of DNA and GoTaq DNA Polymerase (Promega, Madison, USA) employed 35 cycles with denaturing at 95°C for 30 s, annealing at 57°C for 40 s and extension at 72°C for 50 s in a total volume of 15 μl.
A total of 2 μl of the PCR amplification product was submitted to the sequencing reaction using the BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Foster City, USA). Twenty-five cycles were performed employing 12 ng of the sense primer IDH1-JM_f TGATGAGAAGAGGGTTGAGGA, with denaturing at 95°C for 30 s, annealing at 57°C for 15 s and extension at 60°C for 240 s. A second round of sequencing analysis was performed using the antisense primer IDH1-JM_r GCAAAATCACATTATTGCCAAC and the sequencing reaction conditions as described above. Sequences were determined using the semi-automated sequencer (ABI 3100 Genetic Analyzer, Applied Biosystems, Foster City) and the Sequence Pilot version 3.1 (JSI-Medisys, Kippenheim, Germany) software.
IDH1 mutant generation and cloning
IDH1 mutants were generated using the site directed mutagenesis method. Therefore, primers for each mutation were created and used on IDH1 wt cDNA in pDONR221 (DKFZ clone repository). Each mutation was confirmed by Sanger sequencing using the same procedure as described for the FFPE samples. The pDONR221 clones were used for all further LR-reactions into the described destination vectors. LR-reactions were performed following the manufacturers protocol (Invitrogen, Carlsbad, USA).
IDH1 protein purification
To purify the different IDH1 proteins, the cDNAs were transferred into pDEST15 (Invitrogen, Carlsbad, USA), an E. coli expression vector containing a N-terminal GST tag (Invitrogen, Carlsbad, USA). The pDEST15 vectors were then transfected into E.coli expression strain KRX (Promega, Madison, USA). The E.coli were then streaked out on lysogeny broth (LB)-plates containing Ampicillin (100 μg/ml, Sigma-Aldrich, St. Luis, USA) and incubated at 37°C overnight. Six clones from each construct were transferred into 6 ml liquid LB containing 100 μg/ml Ampicillin and grown over night at 37°C in an orbital shaker (220 rpm). From these overnight cultures we used aliquots to prepare two small cultures (10 ml) for induction experiments. We only induced one culture from each colony following the manufacturer’s protocol (KRX protocol, Promega, Madison, USA). Briefly, inoculation of 1:100 of overnight culture, two hours incubation at 37°C and 220 rpm in the orbital shaker followed by the induction with 0.1% L-Rhamnose (10% stock solution in water, AppliChem, Gatersleben, Germany) and four hours incubation at RT and 220 rpm. Subsequently both cultures of each clone were harvested by centrifugation and resuspendet in GST-Lysis buffer from the Pierce® GST Spin Purification Kit (Thermo Scientific, Rockford, USA). Then they went through three freeze-thaw cycles with liquid nitrogen and a 15 min sonification step, followed by a 15 min centrifugation at 5000 rpm on 4°C. The supernatant was used for a SDS-PAGE (Invitrogen, Carlsbad, USA). A coomassie stain of the gel showed us which colonies yield best results. We then used these clones according to the protocol described above to prepare 200 ml cultures. The supernatant of these cultures were then used for protein purification with the Pierce® GST Spin Purification Kit (Thermo Scientific, Rockford, USA), following the manufacturer’s protocol.
IDH1 enzyme kinetics
The measurement of the enzyme kinetics was performed with an Omega FluoStar (BMG Labtech, Ortenberg, Germany) equipped with a pump system, which was used to start the reaction, by adding 4 μg protein to the reaction mixture. All measurements were performed in 96-well plates (BD Falcon, Franklin Lakes, USA) in a total volume of 100 μl at 37°C. The reaction mixture consisted of Tris–HCl pH 7.4 (50 mM), MgCl2 (2 mM), NaCl (10 mM), BSA (0.05%), NADP+/NAPDH (10 mM or concentration row), and isocitrate/α-KG (10 mM or concentration row). As a negative control we used the same mixture, but without isocitrate or α-KG, depending on the KM to be obtained. After the start of the reaction, a one second double orbital shaking (500 rpm) was used to mix the reaction. Thereafter, every two minutes data was obtained from each well measuring the NADPH production or consumption (Ex. 340 +/- 10 nm, Em. 440 +/- 10 nm), depending on the reaction analyzed. All measurements were done in triplicate and at least 15 data points of each reaction were used for KM calculation. The mean of three independent measurements and the standard deviation of these is plotted for each KM.
D-2-hydroxyglutarate
The 2-HG detection was performed with an enzymatic assay developed in our lab. Probe preparations and measurements were performed as described in[16].
D-2-hydroxyglutarate was obtained from Sigma Aldrich (St. Luis, USA) as disodium salt (catalogue number H8378).
Octyl-D-2-hydroxyglutarate was synthesized following the method reported by Xu et al.[12] with slight modifications (detailed protocol for D-2-hydroxyglutarate synthesis, see Additional file1 and Additional file2: Figure S1).
Cell lines
All cell lines were achieved from the ATCC and cultured under standard culture conditions (37°C, 5% CO2) in DMEM medium with 1% Penicillin and Streptomycin and 10% fetal calf serum (all obtained from Gibco® Invitrogen, Carlsbad, USA).
For the generation of IDH1R132H overexpressing cells, IDH1R132H in the destination vector pDEST26 (N-terminal 6x His Tag) was used. LN229 cells were transfected with IDH1R132H in pDEST26 vector by Fugene 6 (Promega, Madison, USA) followed by picking of single cell clones. Single cell clones were selected with 2 mg/ml Gentamycin (Invitrogen, Carlsbad, USA). Clones that survived selection were analyzed for their expression of IDH1R132H by western blot. Clones H3 and H114 were chosen for further analysis, due to their different and stable expression levels of IDH1R132H.
For all experiments with the inducible expression system we used normal cell culture medium, but exchanged normal FCS with 10% Tet system approved FBS (Clontech, Mountain View, USA). To generate an inducible cell line we used the pT-REx-DEST system (Invitrogen, Carlsbad, USA). As a first step we transfected the cell line LN319 with pcDNA6/TR. From this transfection we generated single cell clones and tested their reliability by introducing EGFP in pT-REx-DEST30. We chose the clone with no GFP expression in tetracycline free media and with the highest expression after induction with 1 μM Doxycycline (Sigma-Aldrich, St. Luis, USA). This clone 09 (K09) was then used for all further experiments.
For the experimental setup we used LN319 K09 transfected with IDH1 wild type (wt), the different IDH1 mutants, and GFP in pT-REx-DEST30. All cell lines were seeded as described under proliferation analysis. For each cell line two triplicates were seeded. One was induced with 1 μg/ml Doxycycline, the other one was treated with the comparable amount of solvent (DMSO, Sigma-Aldrich, St. Luis, USA).
To generate cell lines which express IDH1 wt and mutant proteins, we used cDNAs in pMXs-GW-IRES-BsdR and transfected them into HEK293T cells with FuGene®. Cells were subsequently put under selection pressure, by adding 4 μg/ml Blasticidine S (Sigma-Aldrich, St. Luis, USA). As control we used GFP in pMXs-GW-IRES-BsdR.
Cell line analysis
For cell number analysis CellTiterGlo (Promega, Madison, USA) was used in 96-well plate format. All cells were seeded at a density of 5,000 cells/well and subsequently treated. Measurements were performed at the indicated time points following the manufacturer’s protocol.
All migration assays were performed with Ibidis Culture-Inserts. In each well of the insert, 50,000 cells were seeded. After 24 h, the cells were treated with 10 μg/ml Mitomycin C (Sigma-Aldrich, St. Luis, USA) for 2 h, to avoid the influence of proliferation in the assay. Afterward, the inserts were removed leaving a gap of 500 μm +/- 50 μm. Cells were washed with PBS once and thereafter normal medium was applied. This medium was subsequently substituted with 2-HG at the given concentrations. The gap was microscopically documented at the start and the indicated time points. The pictures were analyzed with TScratch (http://chaton.ethz.ch/software/) and the area closed after the indicated time point was plotted in the graph.
The soft agar assays were performed in 6-well plates. The wells were prepared with a 1% agar solution (Agar noble, US Biological, Salem, USA) as base agar. On top of this, 5,000 cells in 0.35% agar were seeded and grown for 14 days. The cells were stained with 0.00025% crystal violet solution and colonies > 1 mm2 were counted.