Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Agonists of retinoid X receptors (RXRs), which include the natural 9-cis-retinoic acid and synthetic analogs, are potent inducers of growth arrest and apoptosis in some cancer cells. As such, they are being used in clinical trials for the treatment and prevention of solid tumors and are used to treat cutaneous T cell lymphoma. However, the molecular mechanisms that underlie the anticancer effects of RXR agonists remain unclear. Here, we show that a novel pro-apoptotic pathway that is induced by RXR agonist is negatively regulated by casein kinase 1␣ (CK1␣). CK1␣ associates with RXR in an agonist-dependent manner and phosphorylates RXR. The ability of an RXR agonist to recruit CK1␣ to a complex with RXR in cells correlates inversely with its ability to inhibit growth. Remarkably, depletion of CK1␣ in resistant cells renders them susceptible to RXR agonist-induced growth inhibition and apoptosis. Our study shows that CK1␣ can promote cell survival by interfering with RXR agonist-induced apoptosis. Inhibition of CK1␣ may enhance the anti-cancer effects of RXR agonists.

Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones. However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes. Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described. In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin. We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors. Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR. These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events.Retinoic acid receptors and retinoid X receptors (RAR and RXR ␣, ␤, and ␥) are sequence-specific, ligand-dependent transcription factors belonging to the superfamily of steroid/thyroid/vitamin D 3 nuclear receptors (1). RAR-RXR 1 heterodimers induce gene expression in a ligand dependent manner through RA responsive elements (RAREs) present in the promoter regions of responsive genes (2). Recently, CBP/p300, Sug1/Trip1, TIF1, SRC-1/N-CoA1, TIF2/GRIP1, and ACTR have been identified as co-factors, which interact with RARs and other nuclear receptors in a ligand-dependent manner (3, 4). Biochemical evidence supports models involving depletion of the nucleosomal core as well as H1 histones at the promoter regions of actively transcribing genes. CBP/p300, its associated protein p/CAF, SRC-1, and ACTR have intrinsic histone acetyltransferase (HAT) activity (5-7). It has been proposed that recruitment of HAT activity by sequence-specific transcription factors leads to acetylation of core histones and a destabilization of the nucleosomal core, thereby facilitating transcriptional activation (8 -10). However, an earlier obligatory step in transcriptional activation involves an unfolding of the compact, 30-nm chromatin fiber, which results only from a displacement of the potent transcriptional repressor, histone H1, from chromatin (11, 12). Mechanisms by which nuclear receptors and other transcription factors affect this obligatory removal of histone H1 from transcriptionally silent chromatin have not been described previously. In this report, we provide evidence that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromati...

Synthetic retinoid-related molecules, such as N-(4-hydroxyphenyl)retinamide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induce apoptosis in a variety of malignant cells. The mechanism(s) of action of these compounds does not appear to involve retinoic acid receptors (RARs) and retinoid X receptors (RXRs), although some investigators disagree with this view. To clarify whether some retinoid-related molecules can induce apoptosis without involving RARs and/or RXRs, we used 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-E-propenyl] benzoic acid (AGN193198) that neither binds effectively to RARs and RXRs nor transactivates in RAR-and RXR-mediated reporter assays. AGN193198 potently induced apoptosis in prostate, breast, and gastrointestinal carcinoma cells and in leukemia cells. AGN193198 also abolished growth (by 50% at 130 -332 nM) and induced apoptosis in primary cultures established from prostatic carcinoma (13 patients) and gastrointestinal carcinoma (1 patient). Apoptosis was induced rapidly, as indicated by mitochondrial depolarization and DNA fragmentation. Molecular events provoked by AGN193198 included activation of caspase-3, -8, -9, and -10 (by 4 -6 h) and the production of BID/p15 (by 6 h). These findings show that caspase-mediated induction of apoptosis by AGN193198 is RAR/RXR-independent and suggest that this compound may be useful in the treatment of prostate cancer.