NF-κB p65 recruited SHP regulates PDCD5-mediated apoptosis in cancer cells.

Transcription factor NF-κB promotes cell proliferation in response to cell injury. Increasing evidence, however, suggests that NF-κB can also play an apoptotic role depending on the stimulus and cell type. We have previously demonstrated that novel retinoid 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated apoptosis in breast carcinoma cells requires activation of canonical and non-canonical NF-κB pathways. The mechanism NF-κB uses to induce apoptosis remains largely unknown. NF-κB subunit p65 (RelA) was identified as one potent transcriptional activator in 3-Cl-AHPC-mediated apoptosis in cells. Here we used ChIP-on-chip to identify NF-κB p65 genes activated in 3-Cl-AHPC mediated apoptosis. This paper focuses on one hit: pro-apoptotic protein programmed cell death 5 (PDCD5). 3-Cl-AHPC mediated apoptosis in MDA-MB-468 had three related effects on PDCD5: NF-κB p65 binding to the PDCD5 gene, enhanced PDCD5 promoter activity, and increased PDCD5 protein expression. Furthermore, 3-Cl-AHPC increased orphan nuclear receptor small heterodimer partner (SHP) mRNA expression, increased SHP protein bound to NF-κB p65, and found the SHP/NF-κB p65 complex attached to the PDCD5 gene. PDCD5 triggered apoptosis through increased Bax protein and release of cytochrome C from mitochondria to cytosol. Lastly, knockdown of PDCD5 protein expression blocked 3-Cl-AHPC mediated apoptosis, while over-expression of PDCD5 enhanced apoptosis, suggesting PDCD5 is necessary and sufficient for NF-κB p65 mediated apoptosis. Our results demonstrate a novel pathway for NF-κB p65 in regulating apoptosis through SHP and PDCD5.

The retinoid X receptors and their ligands.

This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, ß, and γ (RXRs, NR2B1-3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand-bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis.

INTRODUCTION: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, ß and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARß are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/ß in favor of RARγ. METHODS: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis. RESULTS: Modulation of the RARα/ß to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death. CONCLUSIONS: The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/ß balance suggests that prevalence of RARγ over-RARα/ß expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.

Conjugation of antisense oligonucleotides to PEGylated carbon nanotubes enables efficient knockdown of PTPN22 in T lymphocytes.

PEGylated-carbon nanotubes (PNTs) were evaluated as nanocarriers of antisense oligonucleotides into T-cells using protein tyrosine phosphatase N22 (PTPN22) as a model target gene. PTPN22 is an important predisposing gene and drug target in type 1 diabetes and several other human autoimmune diseases. Here, we generated the first anti-PTPN22 20-mer antisense oligonucleotides (ASOs) and tethered them to PNTs through a cleavable disulfide bond. Spectroscopic and atomic force microscopy analyses were used to determine the loading of ASO onto PNTs, whereas the cleavable nature of the disulfide bond connecting the oligonucleotide to the nanocarrier was confirmed by incubation with dithiothreitol followed by agarose gel electrophoresis. PNT-conjugated ASOs achieved efficient (>50%) knockdown of PTPN22 expression in T-lymphocytes in culture at the mRNA and protein level, as measured by quantitative real-time PCR and Western blotting, respectively. Considering the high biocompatibility and low in vivo toxicity of PNTs, we expect that our approach will be easily translated to achieve in vivo knockdown of PTPN22 and other T lymphocyte targets, thus enabling novel ASO-mediated immunotherapies for type 1 diabetes and other autoimmune diseases.

3D-QSAR and QSSR studies of 3,8-diazabicyclo[4.2.0]octane derivatives as neuronal nicotinic acetylcholine receptors by comparative molecular field analysis (CoMFA).

High subtype selectivity (alpha4beta2 over alpha2beta3) of neuronal nicotinic acetylcholine receptor (nAChR) agonists is critical for the rational design of less toxic drugs used for the treatment of neurodegenerative and psychiatric diseases. Here, three CoMFA models of pEC(50)(alpha4beta2), pEC(50)(alpha2beta3) and p[EC(50)(alpha4beta2)/EC(50)(alpha2beta3)] (pEC(50)(alpha4beta2)pEC(50)(alpha2beta3)) were developed to study the quantitative structure-activity relationship (QSAR) and quantitative structure-selectivity relationship (QSSR) of the 3,8-diazabicyclo[4.2.0]octane derivatives as nAChRs agonists. The parameters of the three models were 0.584, 0.792, and 0.599 for cross-validated r(2) (r(2)(CV)), 0.924, 0.935 and 0.875 for conventional r(2). Analyses indicated that both the steric and electrostatic factors should be considered in the rational design of more active and selective nAChR agonists.

Studies of cannabinoid-1 receptor antagonists for the treatment of obesity: hologram QSAR model for biarylpyrazolyl oxadiazole ligands.

Hologram QSAR studies were conducted on a series of 60 training set of cannabinoid-1 receptor (CB(1)) antagonists. Significant cross-validated correlation coefficients (q(2)=0.763) and noncross-validated correlation coefficients (r(2)=0.897) were obtained. The model was then employed to predict the biological activities of 15 test set compounds, and a good agreement between the experimental and predicted values was verified exhibiting a powerful predictable capability of this model (q(pred)(2)=0.868). Contribution map shows that 1,2,4-trizole and cyclopropane moieties make big contributions for the activities. Both the HQSAR model and analysis from the contribution map should be useful for the further design of novel structurally related CB(1) antagonists.

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand-binding domain.

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand-binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRalpha LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator-activated receptor gamma (PPARgamma) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line-broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo- or hetero-dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor-interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRalpha LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C-terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line-broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRalpha LBD-antagonist-peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRalpha LBD conformations in favor of a compact state, and that a PPARgamma LBD-agonist complex can prevent the antagonist from enhancing the RXRalpha LBD-coactivator binding interaction.

Adamantyl-substituted retinoid-related molecules bind small heterodimer partner and modulate the Sin3A repressor.

6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437/AHPN) and 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) are inducers of apoptosis of malignant cells both in vitro and in vivo. Numerous mechanisms have been proposed for how these compounds exert this effect. This report shows that AHPN/3-Cl-AHPC binds specifically to the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), and this binding promotes interaction of the receptor with a corepressor complex that minimally contains Sin3A, N-CoR, histone deacetylase 4, and HSP90. Formation of the SHP-Sin3A complex is essential for the ability of AHPN and 3-Cl-AHPC to induce apoptosis, as both knockout SHP and knockdown of Sin3A compromise the proapoptotic activity of these compounds but not other apoptosis inducers. These results suggest that AHPN/3-Cl-AHPC and their analogues are SHP ligands and their induction of apoptosis is mediated by their binding to the SHP receptor.

Retinoic acid (RA) regulates 17beta-hydroxysteroid dehydrogenase type 2 expression in endometrium: interaction of RA receptors with specificity protein (SP) 1/SP3 for estradiol metabolism.

CONTEXT: The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (HSD17B2) exerts a local antiestrogenic effect by metabolizing biologically active estradiol to inactive estrone in endometrial epithelial cells. Retinoic acid (RA) induces HSD17B2 expression, but the underlying mechanism is not known. OBJECTIVE: Our objective was to elucidate the molecular mechanisms responsible for HSD17B2 expression in human endometrial cells. METHOD: Human endometrial Ishikawa and RL95-2 cell lines were cultured in the presence or absence of RA to analyze endogenous HSD17B2 expression, transcription factor complex formation, and promoter activity. RESULTS: RA induced HSD17B2 mRNA levels in a dose- and time-dependent manner in endometrial cells. The RA antagonist ANG11273 abolished RA-induced HSD17B2 expression. Small interfering RNA ablation of RA receptor (RAR)alpha or retinoid X receptor (RXR)alpha completely blocked RA-induced HSD17B2 gene expression. Analysis of serial deletion and site-directed mutants of the HSD17B2 promoter fused to a reporter gene indicated that RA induction requires a cis-regulatory sequence that binds the specificity protein (SP) class of transcription factors. Chromatin-immunoprecipitation-PCR and gel-shift assays showed that RARalpha/RXRalpha and SP1/SP3 interact with this HSD17B2 promoter sequence. Small interfering RNA ablation of SP1 and SP3 expression markedly decreased HSD17B2 basal expression and blocked RA-induced expression. Finally, immunoprecipitationimmunoblotting demonstrated RA-induced interactions between RARalpha/RXRalpha and SP1/SP3 in intact endometrial cells. CONCLUSIONS: In endometrial epithelial cells, RA stimulates formation of a multimeric complex comprised of RARalpha/RXRalpha tethered to transcription factors SP1 and SP3 on the HSD17B2 promoter. Assembly of this transcriptional complex is necessary for RA induction of HSD17B2 expression and may be an important mechanism for local estradiol inactivation in the endometrium.

Cell-type specific and cytoplasmic targeting of PEGylated carbon nanotube-based nanoassemblies.

In this paper we report the fabrication of a multivalent, cell-type specific and cytoplasmic delivery system based on single-walled carbon nanotubes. The latter were functionalized through adsorption of phospholipids terminated by biotinylated PEG chains functionalized with fluorochrome-coupled neutravidin, and subsequently with antibodies (anti-CD3epsilon and anti-CD28) for T cell receptor post-signaling endocytosis and a synthetic fusogenic polymer for disruption of lysosomal compartments. The biomimetic nanoassemblies were composed by PEGylated individual/very small bundles of carbon nanotubes having an average length and a standard deviation of 176 nm and 77 nm, respectively. The nanoassemblies were stably dispersed under physiological conditions, visible by conventional optical and confocal microscopy and specifically targeted to T cells both in vitro and in living animals. The addition of a fusogenic polymer to the nanoassemblies did not affect the cellular uptake and allowed the release into the cytosol of the targeted cells both in vitro and in the animals. The present manuscript is the first report about the cytoplasmic delivery of carbon nanotubes in a specific cell type in intact animals and paves the way for their use as in vivo intracellular delivery systems.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

An adamantyl-substituted retinoid-derived molecule that inhibits cancer cell growth and angiogenesis by inducing apoptosis and binds to small heterodimer partner nuclear receptor: effects of modifying its carboxylate group on apoptosis, proliferation, and protein-tyrosine phosphatase activity.

Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde, and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorophenyltetrazole, (2E)-5-{2-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]ethenyl}-1H-tetrazole, 5-{4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorobenzylidene}thiazolidine-2,4-dione, and (3E)-4-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]-2-oxobut-3-enal were very modest inhibitors of KG-1 proliferation. The other analogues were minimal inhibitors. Fragment-based QSAR analyses relating the polar termini with cancer cell growth inhibition revealed that length and van der Waals electrostatic surface potential were the most influential features on activity. 3-Cl-AHPC and the 3-chlorophenyltetrazole and 3-chlorobenzylidenethiazolidine-2,4-dione analogues were also able to inhibit SHP-2 protein-tyrosine phosphatase, which is elevated in some leukemias. 3-Cl-AHPC at 1.0 microM induced human microvascular endothelial cell apoptosis but did not inhibit cell migration or tube formation.

Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells.

TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.

Retinoid X receptor regulates Nur77/TR3-dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting.

Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways by acting as a ubiquitous heterodimerization partner of many nuclear receptors, including the orphan receptor Nur77 (also known as TR3 [corrected] or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXRalpha is required for nuclear export and mitochondrial targeting of Nur77 through their unique heterodimerization that is mediated by dimerization interfaces located in their DNA-binding domain. The effects of RXRalpha are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXRalpha ligands suppress NES activity by inducing RXRalpha homodimerization or altering RXRalpha/Nur77 heterodimerization. The RXRalpha NES is also silenced by RXRalpha heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXRalpha/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXRalpha and its involvement in the regulation of the Nur77-dependent apoptotic pathway [corrected]

Noncovalently silylated carbon nanotubes decorated with quantum dots.

A nanoassembly of single-walled carbon nanotubes coated by a thin layer of silica followed by quantum dots was prepared. That the quantum dots retained their photoluminescent properties after deposition onto the silylated carbon nanotubes suggests that the thin layer of silica prevented the quenching of the fluorescence by the nanotubes. This fluorescent nanoassembly represents an excellent building block for photoelectric and optical devices and biological nanoprobes.

Apoptosis induction by a novel retinoid-related molecule requires nuclear factor-kappaB activation.

Nuclear factor-kappaB (NF-kappaB) activation has been shown to be both antiapoptotic and proapoptotic depending on the stimulus and the specific cell type involved. NF-kappaB activation has also been shown to be essential for apoptosis induction by a number of agents. The novel retinoid-related molecule 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) activates NF-kappaB with subsequent apoptosis in a number of cell types. We have found that NF-kappaB activation is essential for 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC activates NF-kappaB through IKKalpha kinase activation and the subsequent degradation of IkappaB alpha. IKKalpha kinase activation is associated with IKKalpha-enhanced binding to HSP90. The HSP90 inhibitor geldanamycin enhances the degradation of IKKalpha and blocks 3-Cl-AHPC activation of NF-kappaB and 3-Cl-AHPC-mediated apoptosis. In addition, inhibition of IkappaB alpha degradation using a dominant-negative IkappaB alpha inhibits 3-Cl-AHPC-mediated apoptosis. NF-kappaB p65 activation is essential for 3-Cl-AHPC apoptosis induction as evidenced by the fact that inhibition of p65 activation utilizing the inhibitor helenalin or loss of p65 expression block 3-Cl-AHPC-mediated apoptosis. NF-kappaB has been shown to be antiapoptotic through its enhanced expression of a number of antiapoptotic proteins including X-linked inhibitor of apoptosis protein (XIAP), c-IAP1, and Bcl-X(L). Whereas exposure to 3-Cl-AHPC results in NF-kappaB activation, it inhibits the expression of XIAP, c-IAP1, and Bcl-X(L) and enhances the expression of proapoptotic molecules, including the death receptors DR4 and DR5 as well as Fas and Rip1. Thus, 3-Cl-AHPC, which is under preclinical development, has pleotrophic effects on malignant cells resulting in their apoptosis.

Investigation of ligand interactions with human RXRalpha by hydrogen/deuterium exchange and mass spectrometry.

Several different agonists of the retinoic X receptor alpha (hRXRalpha) were examined for their effects on the amide H/D exchange kinetics of the homodimeric protein using mass spectrometry. Some agonists, LG 100268, SR11246, and DHA, bind such that slower deuterium exchange-in occurs compared with 9-cis-retinoic acid (9-cis-RA), whereas others, fenretinide and methoprenic acid, result in poorer protection during binding and hence faster exchange-in. Protection against H/D exchange by different agonists and the inhibition of H/D exchange kinetics relative to 9-cis-RA varies markedly in different regions of the protein. Agonists LG 100268, SR11246, and DHA generally inhibit faster exchange processes in the ligand binding regions of hRXRalpha than does the native ligand 9-cis-RA. In at least half of these regions, the level of protection by 9-cis-RA lags behind the agonists even after 60 min. Methoprenic acid did not significantly protect hRXRalpha against amide hydrogen exchange. An efficient method is described for comparing the effects of different agonists on the protein structure of the agonist-RXRalpha complex.

Isolation and characterization of fluorescent nanoparticles from pristine and oxidized electric arc-produced single-walled carbon nanotubes.

Fluorescent nanoparticles were isolated from both pristine and nitric acid-oxidized commercially available carbon nanotubes that had been produced by an electric arc method. The pristine and oxidized carbon nanotube-derived fluorescent nanoparticles exhibited a molecular-weight-dependent photoluminescence in the violet-blue and blue to yellowish-green ranges, respectively. The molecular weight dependency of the photoluminescence was strongly related to the specific supplier. We analyzed the composition and morphology of the fluorescent nanoparticles derived from pristine and oxidized nanotubes from one supplier. We found that the isolated fluorescent materials were mainly composed of calcium and zinc. Moreover, the pristine carbon nanotube-derived fluorescent nanoparticles were hydrophobic and had a narrow distribution of maximal lateral dimension. In contrast, the oxidized carbon nanotube-derived fluorescent nanoparticles were superficially oxidized and/or coated by a thin carbon layer, had the ability to aggregate when dispersed in water, and exhibited a broader distribution of maximal lateral dimension.

Synthesis and characterization of supramolecular nanostructures of carbon nanotubes and ruthenium-complex Luminophores.

We constructed and characterized supramolecular nanostructures consisting of ruthenium-complex luminophores, which were directly grafted onto short oxidized single-walled carbon nanotubes or physically entrapped in silica nanobeads, which had been covalently linked to short oxidized single-walled carbon nanotubes or hydrophobically adsorbed onto full-length multi-walled carbon nanotubes. These structures were evaluated as potential electron-acceptor complexes for use in the fabrication of photovoltaic devices, and for their properties as fluorescent nanocomposites for use in biosensors or nanoelectronics.

Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells.

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.