The pan-RAR agonist all-trans retinoic acid (Sigma) was dissolved in 70% ethanol, to obtain a 1 mM stock solution, while the pan-RAR antagonist AGN193109 (Allergen pharmaceuticals) and MG132 (Calbiochem) were reconstituted in DMSO to obtain a 10 mM stock solution.
Cell lines and cell culture
Wild type Line 1 tumor cells (WT) and their non-malignant counterparts (mIκB), transduced with a dominant negative inhibitor of NFκB were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and maintained at 37°C in a 95% humid atmosphere, with 5% CO2.
Expression profiling of retinoid acid receptors (RARs)
RT-PCR was used to assess the expression levels of retinoid receptor subtypes in WT and mIκB-Line1 tumor cells. 1 μg of total RNA obtained from cell lines was reversed transcribed and amplified for RAR subtypes using the Advantage rapid RT-PCR kit (Promega®), under the following conditions: reverse transcription at 48°C for 60 min, initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturing at 95°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min; followed by a final extension of 72°C for 7 min, in a Stratagene RoboCycler™ Gradient 96 thermal cycler (Stratagene, La Jolla, CA) RAR subtype specific primers used were – RARα (5-ATGTAAGGGCTTCTTCCG-3 & 3-AGTCTTAATGATGCACTT-5), RARβ(5-CTGGCTTGTCTGTCATAATTCA-3 & 3-GGTACTCTGTGTCTCGATGGAT-5), RARγ (5-GTGGAGACCGAATGGACC-3 & 3-GACAGGGATGAACACAGG-5), The expression levels of β-actin (5-GAGCTATGAGCTGCCTGACG-3 & 3-AGCACTTGCGGTGCACGATG-5) and RelA (5-GAAGAAGCGAGACCTGGAGCAA-3 & 3-GTTGATGGTGCTGAGGGATGCT-5) were assessed under identical conditions.
Assessment of differential DNA binding and transcriptional activity of RARs in WT and mIκB-Line 1 tumor cells
Electromobility shift assay was used to contrast RAR-DNA binding activity in WT and mIκB-Line1 cells. Briefly, 5 μg of nuclear extracts were admixed with 2 μg of poly (di-dc) and DNA binding buffer (50 mM NaCl, 5 mM HEPES (pH 7.5), 5 mM EDTA, 10% EGTA, 30% glycerol and 1.25 μg BSA) in a total volume of 10 μl and incubated on ice for 20 min. RAR and RXR oligonucleotides (Santa Cruz Biotech) were end labeled by use of T4 polynucleotide kinase and [32P] cytosine triphosphate (DuPont NEN) and 20,000 cpm of the 32P labeled oligonucleotides added to the binding reaction and incubated for 30 min at room temperature. The complexes were subsequently separated on a 6% polyacrylamide gel under non-denaturing conditions at 125 Volts for 3 h. Gels were dried on 3 M Whatman papers and the DNA-protein complexes visualized by autoradiography.
RAR and NFκB transcriptional activity was assessed by transient transfection of 0.8 μg of pRAR-firefly luciferase construct (trimerized retinoic acid receptor-beta 2 response element, generously provided by Dr M.T Underhill) or pNFκB-firefly luciferase construct (Promega) plus 2 ng of pRL-SV40 (Promega) renilla luciferase to normalize and control for tranfection efficiencies. Plasmids were incubated with 3 μl of Lipofectamine 2000 (Gibco) in serum free DMEM for 15 min, and the complex added to 70% confluent well of a 6 well plate. Experiments were performed in triplicates, and the transfection reagents scaled up accordingly.
Physical association of RARs to NFκB-DNA complexes and the ligand responsiveness of these interactions
WT-Line1 tumor cells were exposed to increasing concentrations of at-RA (0.1–1 μM) or increasing concentrations of AGN193109 (1–10 μM) in the presence of 1 μM at-RA for 24 h. Cells thus treated were re-suspended in 1 ml of ice cold RIPA buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 1 μM PMSF and 1 μM DTT), incubated on ice for 30 min and resulting suspension pelleted by centrifugation at 14000 rpms for 10 minutes. 200 μls of the supernatant thus obtained was added to 100 μls of NFκB-oligonucleotide agarose conjugate slurry (Santa Cruz), plus 300 μls of binding buffer (10 mM Tris, pH 7.5; 50 mM NaCl; 1 mM DTT; 1 mM EDTA; 5% glycerol; 1 μg/ml poly dI-dC), and incubated overnight at 4°C with constant rocking. After the overnight incubation, agarose beads were washed thrice in binding buffer, re-suspended in 30 μl of protein loading dye, boiled for 5 min and analyzed by western blot analysis. RAR or NFκB–RelA antibodies (Santa Cruz) were used in conjunction with protein A-peroxidase conjugate and immunoreactive bands were detected using the enhanced chemiluminescence system (Amersham) after exposure to Hyperfilm ECL (Amersham). 5 μg of cell lysates were equally analyzed by western blot, for changes in the expression level of RAR and p-65 NFκB following the 24 h drug exposure.
Analysis of pro-metastastic MMP 9 and anti-metastatic TIMP 1 gene expression in response to at-RA and AGN193109 by real-time PCR
Total RNA was extracted from control and drug exposed cells using Quaigen RNAeasy miniprep columns following the manufacturers recommendations. Total RNA thus obtained was quantified by UV absorption at 260/280 λ (Genequant), and subjected to Northern blot analysis for the expression of MMP9 and TIMP1 as previously described. To enhance sensitivity, we utilized real time PCR analysis to appreciate changes in RAR, RelA, MMP9 and TIMP1 message levels. Briefly, 1 μg of RNA was reversed transcribed and diluted 5 fold in RNAse free water. 2 μl of the cDNA thus obtained was PCR amplified in a mix of 18 μl PCR supermix (GibcoBrl) plus the 2 μl of fluorescent DNA intercalating dye SYBR green (1:3000) using the real time PCR machine (Rotor-Gene 2000 Robocycler, Phenix research).
PCR primer pairs for MMP 9 were: 5'-TGAAACCAGACCCCAGACTC-3' and 5'-TGA ACC ATA ACG CAC AGA CC-3' and for TIMP 1 were: 5'-ATG CCC ACA AGT CCC AGA AC-3' and 5'-TACGCCAGGGAACCAAGAAG-3' and the PCR conditions were: Initial denaturing at 95°C for 2 min, followed by 40 cycles of 95°C denaturing for 45 s, 60°C annealing for 1 min and 72°C extension for 1 min.
Experiments were performed in triplicates and results are expressed as a standard error of the mean. Statistical analyses were done using the student t-test and ANOVA.