Cell lines and reagents.
COS7, PANC1, and SKNAS cells were grown in DMEM with 10% FBS. All ESFT cell lines, DAOY, D556, and H1299 were grown in RPMI with 10% FBS and 1% HEPES, with the exception of SKES and A673. SKES cells were grown in McCoy’s 5a medium with 15% FBS. A673 cells were grown in DMEM supplemented with 1% sodium pyruvate and 10% FBS. CAPAN cells were grown in Iscove DMEM (IMDM) with 1% sodium pyruvate and 20% FBS. CHLA-20 and CHLA-15 cells were grown in IMDM with 1% insulin-transferrin-sodium selenate and 20% FBS. HepG2 cells were grown in DMEM with 10% FBS and 1% nonessential amino acids. KIF3a WT and knockout MEFS were grown in DMEM plus 1% nonessential amino acids, 1% l-glutamine, 1% sodium pyruvate, and G418 (mg/ml). Pharmaceutical-grade ATO was purchased at a concentration of 1 mg/ml in a sterile, nonpyrogenic, clear solution in water using sodium hydroxide and dilute hydrochloric acid to adjust to pH 8 (Cephalon Inc.). The JNK inhibitor SP600125 was purchased from Calbiochem. LPS (Sigma-Aldrich) was used to activate NF-κB.
GLI1 activity was assessed using a pGL3–basic luciferase construct containing 8× GLI1-binding sites attached to a chicken lens crystalline promoter (pGL38xGLI; ATCC–Johns Hopkins Special Collections, deposited by P.A. Beachy) and a Renilla-TK mutant construct (provided by S. Byers, Georgetown University, Washington, DC, USA). To measure the effects of ATO on GLI1, HepG2 cells were cotransfected with either EGFP-hGLI1 (provided by J. Reiter, UCSF, San Francisco, California, USA) or empty vector control (pEGFP-C1; Clontech) in addition to pGL38xGLI luciferase and Renilla constructs with Fugene6 (Roche) according the manufacturer’s protocol. To measure the effects of ATO on other transcription factors, HEPG2 cells were cotransfected with EWS-FLI1 and the EWS-FLI1–responsive luciferase constructs NROB1 (provided by S. Lessnick, University of Utah, Salt Lake City, Utah, USA) or Notch1 (provided by R. Schlegel, Georgetown University, Washington, DC, USA) and the Notch-responsive promoter constructor HES-1 (provided by A. Israel, Institut Pasteur, Paris, France) and empty vector control construct (CIneo or pFlagCMV2) in addition to the Renilla construct. To measure the effects of GLI2, COS7 cells were transfected with or without pcDNA4T/O-GLI2 (provided by F. Aberger, University of Salzberg, Salzberg, Austria) in addition to pGL38xGLI luciferase and Renilla constructs with Fugene6. To measure the effects of ATO on GLI1 activity in the nucleus, we transfected COS7 cells with EGFP-GLI1WT, EGFP-GLI1AHA (provided by D. Wu, Yale University, New Haven, Connecticut, USA), and EGFP-GLI1WT cotransfected with HA-tagged DRYK1 (provided by D. Wu) in addition to the pGL3-8xGLI1 luciferase and Renilla constructs. Localization was determined by green fluorescence for GFP-tagged proteins and Hoechst dye for the nucleus on a Nikon Eclipse Ti microscope using a ×40 lens. In studies using drug treatment, cells were treated with drug 24 hours after transfection, and then luciferase activity was measured 24 hours after treatment. Luciferase assays in the KIF3a WT and knockout cells were done similar to those described above; however, the MEFs were transfected using electroporation in OptiMEM medium (Invitrogen) using a Cell-Porator (Invitrogen) at 350 V. To measure the effect of JNK inhibition in combination with ATO treatment on GLI1 activity, COS7 cells were transfected with EGFP-hGLI1 in addition to pGL38xGLI luciferase and Renilla constructs with Fugene6 (Roche) according the manufacturer’s protocol. 24 hours after transfection, cells were drug treated; luciferase activity was measured 24 hours after treatment. All luciferase assays were performed using a dual luciferase assay kit according the manufacturer’s protocol (Promega).
Immunofluorescence for visualization of primary cilia.
KIF3a WT or knockout cells were plated on circular coverslips in 12-well plates and grown to confluency. Cells were then serum starved for 24 hours to form primary cilia, treated with ATO for another 24 hours in the absence of serum, and then fixed in 4% paraformaldehyde for 20 minutes. Cells were permeabilized with PBS with 0.5% Triton X-100 for 5 minutes, and nonspecific binding sites were blocked with 2% BSA in PBST (PBS with 0.5% Tween-20). Cells were stained with primary antibody for acetylated tubulin (Sigma-Aldrich, 1:5,000) diluted in 2% BSA in PBST overnight at 4°C. After washing 3 times in PBST, cells were incubated for 1 hour with Alexa Fluor 488–conjugated secondary antibodies (Invitrogen) in 2% BSA in PBST. Cilia formation was then viewed using an Olympus Fluoview-FV300 Laser Scanning Confocal System.
Cloning and protein expression of recombinant full-length GLI1.
GLI1 cDNA was isolated from the EGFP-hGLI1 using PCR with the following primers: forward, 5′-TTCAACTCGATGACCCCACC-3′; reverse, 5′-GGCACTAGAGTTGAGGAATT-3′. The forward primer added an ATG start site, and the reverse removed the stop codon. The PCR product was then TA cloned into the pCRII vector (Invitrogen) according to the manufacturer’s instructions. The N-terminal HIS and T7 tags were removed from pET28B+ (Novagen; EMD Biosciences Inc.) by restriction digest with NcoI and BamHI followed by mungbean endonuclease treatment to make blunt ends. The blunt ends were ligated together using T4 DNA ligase (Roche) according to the manufacturer’s instructions. In order to have the C-terminal HIS tag in frame with the GLI1 sequence, both the modified pET28B+ and GLI1 in TA vector pCRII were first cut with NotI, and then the overhang was filled in with DNA polymerase I–klenow fragment (New England Biolabs) according to the manufacturer’s protocol to make a blunt end. Both were cut with SacI, and the GLI1 sequence was ligated to the pET28B+ using T4 DNA ligase. The resulting vector pET28B+/GLI1 was verified by sequencing. BL21-CodonPlus (DE3)–RIPL-competent cells (Stratagene) containing the expression vector were grown overnight at 37°C in Luria-Bertani media with 50 μg/ml kanamycin and chloramphenicol. The culture was then diluted 1:20 in 1 l fresh Luria-Bertani media without antibiotics and allowed to grow at 37°C until an optical density of 0.6 was observed at 600 nm. Protein production was initiated by adding 1 mM isopropyl-D-1-thiogalactopyranoside, and bacteria were cultured for an additional 4 hours at 37°C. The majority of the protein was found to be in the insoluble inclusion body fraction of the bacteria. The insoluble and soluble fractions were separated and isolated from the whole bacteria prep with BugBuster protein extraction reagent (Novagen) according to the manufacturer’s protocol.
Recombinant GLI1 protein preparation.
An inclusion body pellet corresponding to 1 l bacterial culture was resuspended in 50 ml of buffer containing 20 mM Tris-HCL (pH 7.5), 500 mM NaCl, 5 mM imidazole, and 6 M guanidine HCL. The supernatant was filtered (0.22 μm pore size) and applied to a 1-ml prepacked Hitrap metal chelating column on an AKTA-Explorer chromatography system kept at 4°C (GE Healthcare). Initially, metal chelating columns were washed with 10 ml H2O; charged with 10 ml of 100 mM NiSO4; washed again with H2O; and equilibrated with 20 mM Tris-HCL (pH 7.5), 500 mM NaCl, 5 mM imidazole, and 6 M guanidine HCL. The sample was loaded on the column, and buffer was changed to 10 ml of 20 mM Tris-HCL (pH 7.5), 500 mM NaCl, 5 mM imidazole, and 6 M urea in a gradient. The protein was then refolded on the column using a slow 30-ml linear gradient that changed to 20 mM Tris-HCL (pH 7.5), 500 mM NaCl, 5 mM imidazole, and 1 mM ZnCl2. Protein was eluted from the column by a slow 20-ml linear gradient from 5 mM to 2 M imidazole. ZnCl2 was added to refolding and elution buffers to help maintain the integrity of the zinc finger DNA binding domains. Aliquots (1 ml) were collected and then applied to a 6% acrylamide gel for Coomassie staining (ThermoScientific).
SPR studies for direct DNA binding.
SPR studies were performed on a Biacore T-100 instrument at room temperature. GLI1 protein was captured on the second flow cell of a CM5 sensorchip by amine coupling method in pH 5.0 glycine buffer (~8,000 RU). The first flow cell was left empty for background signal subtraction. All DNA oligonucleotides were diluted in the running buffer (10 mM HEPES, pH 7.4; 90 mM KCl2; 1 mM ZnCl2; 0.5 mM EDTA; and 0.05% p-20). Kinetics analysis was done by injecting WT (forward, 5′-AGCTACCTGGGTGGTCTCT-3′; reverse, 5′-AGAGACCACCCAGGTAGCT-3′) and mutant (forward, 5′-AGCTACCTCCCACTTCTCT-3′; reverse, 5′-AGAGAAGTGGGAGGTAGT-3′) oligonucleotides over GLI1 captured and control surfaces. Each injection was done with 60 seconds association time and 360 seconds dissociation time. For surface regeneration, a 30-second pulse of 1 M NaCl was used. Each concentration was repeated 3 times, and the results were analyzed using Biacore T-100 evaluation software. Experiments were repeated at least 3 times. For reciprocal experiments, the same GLI binding site oligos were synthesized with a Biotin molecule on the 5′ end of 1 strand and captured on a streptavidin-coated chip.
In vitro FlAsH-binding assays.
In vitro FlAsH-EDT2
–binding assays (Invitrogen) were done similar to previously published methods (21
). Briefly, 40 nM recombinant full-length GLI1 was preincubated with 200, 100, 50, and 25 μM ATO for 2 hours in 1× PBS. After 2 hours, 200 nM FlAsH compound was added and incubated for 20 minutes. Recombinant EWS-FLI1 with FlAsH alone was used as a negative control. 50 μM BAL (Invitrogen) was used a positive control, as it quenches FlAsH by directly binding to it. Fluorescence signal was measured with excitation at 508 nm and emission at 528 nm in a PerkinElmer EnVision Multilabel Plate Reader.
ReAsH-EDT2 labeling in cells.
COS7 cells expressing EGFP-fused constructs were grown on cover glasses and labeled with 2.5 μM ReAsH-EDT2 for 1 hour at 37°C in serum-free Opti-MEM (Invitrogen). After thoroughly washing with 250 μM BAL buffer, cells were then live imaged using an Olympus Fluoview-FV300 Laser Scanning Confocal System.
Cells were rinsed 3 times with cold 1× PBS, subsequently lysed in cold hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, and 0.2 mM magnesium chloride), collected, and homogenized with 35 strokes of a glass dounce homogenizer. Homogenates were centrifuged for 10 minutes at 3,000 g, and the supernatant was transferred to an ultracentrifuge tube containing a 1.25 M sucrose pad and protease inhibitors (250 mM sodium fluoride, 5 mM sodium orthovanadate, 5× mini complete protease cocktail [Roche], and 5 mM EDTA). This preparation was centrifuged for 1 hour at 150,000 g (Beckman L8-M ultracentrifuge); protein from the resulting surpernatant was precipitated with 100% ethanol and centrifuged again for 1 hour at 150,000 g; and the resulting pellet, designated as the cytoplasmic pool, was resuspended in SDS sample buffer (2% SDS; 60 mM Tris-HCl, pH 6.8; and 10% glycerol). The pellet from the original centrifugation (nuclei and membrane) was rinsed in hypotonic lysis buffer, recentrifuged, and dried briefly at 4°C. This pellet was resuspended in NP-40 buffer with protease inhibitors (1% NP-40; 150 mM sodium chloride; and 50 mM Tris-HCl, pH 8.0; with 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 1× mini complete protease inhibitor cocktail), briefly vortexed, and allowed to solubilize at 4°C with gentle agitation. The suspension was collected by centrifugation at 3,000 g, resulting in a supernatant (membrane pool) and a pellet (nuclear pool). The pellet was rinsed once in fresh NP-40 buffer and subsequently resuspended in SDS sample buffer.
Whole cell lysates from cells grown to near confluency or tumor samples were subject to SDS-PAGE and then transferred to an Immobilon-P membrane (Millipore). Membranes were then subjected to blocking in 5% nonfat dry milk in 1× TTBS (20 mM Tris-HCL, pH 7.5; 150 mM NaCl; and 0.5% Tween 20) for 1 hour. Dilutions for primary antibodies were as follows: anti-GLI1 (L42B10) for human GLI1 at 1:1,000 (Cell Signaling), anti-GLI1 (V812) for endogenous Cos7 GLI1 at 1:1,000 (Cell Signaling), anti-GLI2 at 1:1,000 (Cell Signaling), anti-FLI1 at 1:2,000 (Santa Cruz Biotechnology), anti-Notch at 1:1,000 (Cell Signaling), anti-actin–HRP (C-11) at 1:3,000 (Santa Cruz Biotechnology), anti–β-tubulin at 1:5,000 (MP Biomedicals), anti-KIF3a at 1:5,000 (Sigma-Aldrich), anti–lamin A/C at 1:2,000 (Cell Signaling), phospho-JNK at 1:1,000 (Cell Signaling), JNK at 1:1,000 (Cell Signaling), phospho–NF-κB p65 at 1:1,000 (Cell Signaling), and NF-κB p65 at 1:1,000 (Cell Signaling). Primary antibodies were added to the membrane in 5% nonfat dry milk in 1× TTBS for 1 hour. The membrane was then washed 3 times in 1× TTBS and HRP-linked anti-rabbit or anti-mouse secondary antibody (GE Healthcare) was added for 1 hour. Blots were washed 3 times in 1× TTBS and then developed using Millipore Immobilon Western Chemiluminescent HRP Substrate per the manufacturer’s instructions (Millipore Corp.). Chemiluminescence was detected using a Fujifilm LAS-3000 imaging system. Densitometry values were obtained using Multigauge software.
For in vitro studies, cells were treated with or without ATO for 12 hours. For in vivo studies, frozen tumors samples were homogenized in liquid nitrogen using a mortar and pestle. Total RNA was extracted by TRIzol (Invitrogen) and reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s protocol. PCR was performed using an Eppendorf Mastercycler realplex using Taqman Gene Expression Master Mix according to the manufacturer’s instructions with Taqman Gene expression primers for GLI1, PTCH1, GAS1, N-myc, and GAPDH (Applied Biosystems). Data were analyzed for expression relative to GAPDH using the comparative Ct method. Each experiment was performed in triplicate.
To determine necrosis, slides were stained with hematoxylin (Harris Modified Hematoxylin; Fisher) at a 1:17 dilution for 2 minutes at room temperature, blued in 1% ammonium hydroxide for 1 minute at room temperature, dehydrated, and mounted with Acrymount. TUNEL assay was performed using the Apotag kit from Millipore. Briefly, 5-μm sections from formalin-fixed, paraffin-embedded tissues were deparaffinized with xylenes and rehydrated through a graded alcohol series. Heat-induced epitope retrieval was performed by immersing the tissue sections at 98°C for 20 minutes in 10 mM citrate buffer (pH 6.0) with 0.05% Tween. Slides were pretreated with 3% hydrogen peroxide at room temperature, 10 mM sodium citrate at 65°C, and equilibration buffer. Slides were exposed to terminal transferase and digoxigenin-labeled dUTP in reaction buffer for 2 hours at 37°C, stopped in wash buffer, and blocked with 10% normal goat serum. Slides were exposed to HRP-conjugated anti-digoxigenin secondary antibodies (Roche) and DAB chromagen (Dako). Quantification of necrosis and TUNEL staining was performed by a pathologist in a blinded fashion with coded samples.
Cellular proliferation assays.
Cellular proliferation was assessed by triplicate plating at a density of 5,000–10,000 cells/well, depending on the cell line, in a 96-well plate. ATO at varying concentrations or vehicle alone (10 μM H2O) were added to cells in standard growth media for that cell line the following morning after plating, once the cells had attached. Fresh media containing drug or vehicle was added every 2–3 days. Viable cells were quantified using 10 μl WST-1 Reagent (Roche) according to the manufacturer’s protocol after 4 days. IC50 values were calculated by sigmoidal dose-response curve fit using Prism Graphpad 4.0 for Macintosh. For Supplemental Figures 7–9, cellular proliferation was assessed using a real-time Electric Cell-substrate Impedance Sensing assay (RTCA DP Analyzer; Roche), and measurements were taken every 10 minutes for 24 hours. Cell Index (CI) is derived as a change in measured electrical impedance to represent cell status. When more cells are attached on the electrodes, the CI values are larger. Thus, CI is a quantitative measure of cell number present in a well. A change in cell status, such as cell morphology, cell adhesion, or cell viability, will lead to a change in CI.
1 × 106
TC-71 cells in 100 μl of HBSS were orthotopically injected into the gastrocnemius muscle of 4-week-old SCID-beige mice (Charles River). We randomized mice to treatment groups receiving every other day i.p. injections of vehicle (PBS) or ATO at a dose of 0.15 mg/kg in 100 μl when tumors were palpable. We measured tumor length and width every 1–2 days and calculated volume with the formula (D
) × π/6, where D
is the longest diameter and d
is the shorter diameter. ND2:SmoA1 homozygous mice were as previously described (42
). Tumor was detected by monitoring for the presence of clinical signs of disease, such as decreased motor and coordination functions, a deformed cranium and megalencephaly, a hunched back, and weight loss. MRI was subsequently performed to confirm the presence of a defined medulloblastoma (MB) tumor in the cerebellum as well as to identify localization and assess volume. Once animals had a detectable tumor, they began treatment with ATO at a dose of 0.15 mg/kg 3 times per week. The end point of the study was when animals were euthanized due to lack of movement around the cage, which resulted in an inability to reach food and water, or weight loss greater than 15% of the total body weight. Animals chosen for Kaplan-Meier analysis between ATO treatment and control were matched based on age of tumor onset as well as tumor size at the start of treatment. All animal studies were approved by the Georgetown University Institutional Animal Care and Use Committee.
All statistical analysis tests were performed using Prism Graphpad 4.0 for Macintosh. Survival curves were analyzed by the Kaplan-Meier method. Correlation was assessed using nonparametric Spearman test. Significance of differences with regard to tumor size was determined by curve regression and F test. IC50 values were calculating using a nonlinear sigmoidal dose-response curve fit. Other determinations of significance were assessed by 2-tailed Student’s t test, as indicated. A P value less than 0.05 was considered significant.