Contrasting RAR transcript levels in WT and mIκB-Line 1 tumor cells by RT-PCR, we demonstrate the induction of all RAR subtypes in mIκB-Line 1 tumor cells (Fig 1A). Although all RAR subtype transcripts are detected, only RARβ protein is detectable and demonstrably enhanced in mIκB-Line 1 tumor cells (Fig 1B). Accordingly, basal RAR reporter activity is five fold induced in mIκB-Line 1 tumor cells, relative to their WT counterparts (Fig 1C).

In WT-Line1 cells, the pan-RAR agonist, at-RA dose dependently activates RAR trans-activation. Using 1 μM at-RA as the optimal dose for induction of RAR reporter activity, co-incubation with the pan-RAR antagonist AGN193109 (1–10 μM), results in a dose-dependent decrease in RAR reporter activity (Fig 2A). Consistent with the inverse antagonistic property of AGN193109 27, 1–10 μM concentrations of AGN193109 alone suppress RAR reporter activity below basal levels (data not shown). However, in the presence of 1 μM at-RA, 10 μM AGN193109 has an agonistic tendency.

The dose-dependent repression of NFκB reporter activity by at-RA and its reversal by AGN193109 (Fig 2B) again verifies the mutually antagonistic interactions between RAR and NFκB. In the presence of 1 μM at-RA, 10 μM AGN193109 is again observed to have an agonistic tendency. To appreciate the basis for these reciprocal signaling schemes, we assessed for RAR and NFκB (p65) interactions on artificial promoter-enhancer elements.

WT-Line1 cells, with high basal NFκB-DNA binding activity, demonstrate considerably lower RAR-DNA binding activity in contrast to mIκB-Line1 tumor cells, with lower basal NFκB-DNA binding activity 9. Activation of NFκB with phorbol myristate acetate 9 results in a decrease in RAR-DNA binding activity in both WT and mIκB-Line1 cells (Fig 3A). These data suggest that NFκB activation (nuclear translocation) precludes RAR-DNA binding. Identical experiments performed in the presence of an RXR DR-1 type oligonucleotide sequence demonstrate no appreciable change in RXR-DNA binding (Fig 3A), although an increase in the expression of RXR subtypes was documented in the mIκB-Line1 cells (data not shown). Furthermore, there is no change in RXR-DNA binding, following induction of NFκB translocation by PMA. Given that RXR is a heterodimeric partner for multiple members of the nuclear hormone receptor superfamily 22, it is conceivable that this assay system is overwhelmed by mass effect.

In our hands, at-RA did not affect NFκB-DNA binding in standard gelshift assays. We note however that ligand activated RXR has been reported to preclude NFκB-DNA binding activity in a cell free system that implicates higher ligand-receptor ratios than otherwise achievable 15. To overcome the limitations of the standard gelshift assay, we utilized a gelshift oligonucleotide pull-down assay (see methods) that allows for the assessment of native protein-DNA interactions at a concentration three orders of magnitude higher than a standard gelshift assay. Using this technique, we demonstrate a dose dependent increase in RAR binding to NFκB-DNA complexes in response to at-RA (Fig 3B), and its reversal by increasing concentrations of AGN193109 (Fig 3B). These results taken together with the reporter experiments, suggest that in the "on" state (the holo receptor conformation), RARs bind to NFκB-DNA complexes and trans-repress NFκB activity while the "off" state (apo receptor conformation) is non-associative, and allows for NFκB trans-activity.

NFκB reciprocally regulates putative pro-metastatic and anti-metastic factors 9. To assess the regulation of pro-metastatic and anti-metastatic gene expression by at-RA and AGN193109, we examined for changes in pro-metastatic MMP 9 and anti-metastic TIMP 1 gene expression. We demonstrate a dose-dependent induction of TIMP 1 and reciprocal repression of MMP 9 gene expression in response to increasing concentrations of at-RA. The effects of at-RA are again mitigated by AGN193109 (Fig 4). We have previously shown, in the same model system, that the net result of the reciprocal regulation of MMP 9 and TIMP 1 gene expression by at-RA is the suppression of MMP 9 activity, tumor cell invasiveness in vitro and spontaneous metastasis in vivo 28. We however exercise caution in speculating on the in vivo effects of AGN193109, given low doses are antagonistic while higher doses have agonistic tendency albeit not intrinsic. A fortiori, high doses of retinoid antagonists have antiproliferative ("agonist-like") activities 29. This underscores the notion of ligand-receptor interactions as "rheostatic" (modulators) and not "static" (agonist/on – antagonist/off) switches.

In keeping with the rheostatic nature of RAR signaling function, at-RA dose-dependently activates RAR signaling while inducing a dose-dependent decrease in RAR protein expression. This pattern is reversed by co-incubation with AGN193109 (Fig 5A). Ligand induced ubiquitination and proteosomal degradation of RARs 2526, is a permissive event and resetting mechanism for RAR trans-activation. Accordingly, RAR trans-activity and degradation is blocked by the proteosome inhibitor MG132 (5 μM) (5B). Although proteosome inhibitors independently inhibit NFκB activity by maintaining inhibitory IκBα protein levels, trans-repression of NFκB by ligand activated RAR is enhanced by MG132, potentially by maintaining RAR protein expression (Fig 5B). The significance of the latter mechanism is asserted by the compound repression of NFκB activity by MG132, on mIκB-Line1 cells expressing a genetically engineered IκBα, not susceptible to proteosomal degradation (Fig 5C).

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.

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.

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.

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.

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.