Metastatic phenotype in CWR22 prostate cancer xenograft following castration.

BACKGROUND: CWR22 is a human xenograft model of primary prostate cancer (PCa) that is often utilized to study castration recurrent (CR) PCa. CWR22 recapitulates clinical response to androgen deprivation therapy (ADT), in that tumors regress in response to castration, but can recur after a period of time. METHODS: Two cohorts of mice, totaling 117 mice were implanted with CWR22, allowed to develop tumors, castrated by pellet removal and followed for a period of 32 and 50 weeks. Mice presenting with tumors >2.0 cm(3) at the primary site, moribund appearance, or palpable masses other than the primary tumor were sacrificed prior to the endpoint of the study. Tumor tissue, serum, and abnormal lesions were collected upon necropsy and analyzed by IHC, H&E, and PCR for presence of metastatic lesions arising from CWR22. RESULTS: Herein, we report that CWR22 progresses after castration from a primary, hormonal therapy-naïve tumor to metastatic disease in 20% of castrated nude mice. Histological examination of CWR22 primary tumors revealed distinct pathologies that correlated with metastatic outcome after castration. CONCLUSION: This is the first report and characterization of spontaneous metastasis in the CWR22 model, thus, CWR22 is a bona-fide model of clinical PCa representing the full progression from androgen-sensitive, primary PCa to metastatic CR-PCa.

Protein kinase Cα signaling regulates inhibitor of DNA binding 1 in the intestinal epithelium.

Increasing evidence supports a role for PKCα in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKCα signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKCα and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKCα activation, and (b) loss of PKCα in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKCα activity in IEC-18 nontransformed intestinal crypt cells determined that PKCα suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKCα, but not PKCδ, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKCα. Notably, Id1 expression was elevated in the intestinal epithelium of PKCα-knock-out mice, confirming that PKCα regulates Id1 in vivo. A wider role for PKCα in control of inhibitor of DNA binding factors is supported by its ability to down-regulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKCα → Erk ⊣ Id1 → cyclin D1 signaling axis in the maintenance of intestinal homeostasis.

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis.

RNA is an important therapeutic target; however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096 member 3 × 3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, druglike benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside-RNA hairpin loop interactions.

Herein, we report the identification of RNA hairpin loops that bind derivatives of kanamycin A, tobramycin, neamine, and neomycin B via two-dimensional combinatorial screening, a method that screens chemical and RNA spaces simultaneously. An arrayed aminoglycoside library was probed for binding to a 6-nucleotide RNA hairpin loop library (4096 members). Members of the loop library that bound each aminoglycoside were excised from the array, amplified and sequenced. Sequences were analyzed with our newly developed RNA Privileged Space Predictor (RNA-PSP) program, which analyzes selected sequences to identify statistically significant trends. RNA-PSP identified the following unique trends: 5'UNNNC3' loops for the kanamycin A derivative (where N is any nucleotide); 5'UNNC3' loops for the tobramycin derivative; 5'UNC3' loops for the neamine derivative; and 5'UNNG3' loops for the neomycin B derivative. The affinities and selectivities of a subset of the ligand-hairpin loop interactions were determined. The selected interactions have K(d) values ranging from 10 nM to 605 nM. Selectivities ranged from 0.4 to >200-fold. Interestingly, the results from RNA-PSP are able to qualitatively predict specificity based on overlap between the RNA sequences selected for the ligands. These studies expand the information available on small molecule-RNA motif interactions, which could be useful to design ligands targeting RNA.

The Privileged Chemical Space Predictor (PCSP): a computer program that identifies privileged chemical space from screens of modularly assembled chemical libraries.

Modularly assembled combinatorial libraries are often used to identify ligands that bind to and modulate the function of a protein or a nucleic acid. Much of the data from screening these compounds, however, is not efficiently utilized to define structure-activity relationships (SAR). If SAR data are accurately constructed, it can enable the design of more potent binders. Herein, we describe a computer program called Privileged Chemical Space Predictor (PCSP) that statistically determines SAR from high-throughput screening (HTS) data and then identifies features in small molecules that predispose them for binding a target. Features are scored for statistical significance and can be utilized to design improved second generation compounds or more target-focused libraries. The program's utility is demonstrated through analysis of a modularly assembled peptoid library that previously was screened for binding to and inhibiting a group I intron RNA from the fungal pathogen Candida albicans.

T1 relaxation measurement with solvent suppression.

Three solvent-suppression pulse sequences of varying complexity were incorporated into the standard inversion recovery pulse program and experimentally evaluated. The least complex suppression sequence involves a composite 90 degrees pulse. A more complex sequence utilizes an excitation sculpting sequence requiring pulsed field gradients, and the most complex sequence incorporates an excitation sculpting sequence with selective rf pulses and gradient pulses. The quality of the spectral data and the accuracy of T(1) measurements of the investigated suppression schemes were evaluated using three aqueous samples with increasing proton content in the water solvent, i.e. by volume 100% D(2)O, 80/20% D(2)O/H(2)O, and 100% H(2)O. For lines removed from the water resonance the T(1) values were generally very consistent between all pulse sequences tested. For lines less than about 200 Hz from the water signal the T(1) measurements become less reliable but are still possible for most of the tested pulse programs.

Hidden Properties of Carbon Dots Revealed After HPLC Fractionation.

Carbon dots (C-dots) are often synthesized, modified, and studied as a mixture. Unfortunately, the spectroscopic and biological properties measured for such C-dots assume that there is a high degree of homogeneity in the produced sample. By means of high-resolution separation techniques, we show that "as-synthesized" C-dots exist as a relatively complex mixture and that an unprecedented reduction in such complexity can reveal fractions of C-dots with unique luminescence properties. The wavelength-dependent photoluminescence commonly assigned as an inherent property of C-dots is not present in fractionated samples. While ultraviolet-visible absorption profiles reported for C-dots are typically featureless, we have found fractions of C-dots possessing unique absorption bands, with different fractions possessing specific emission wavelengths. Furthermore, fractionated C-dots showed profound differences in emission quantum yield, allowing for brighter C-dots to be isolated from an apparent low quantum yield mixture. These more luminescent fractions of C-dots displayed improved biological compatibility and usefulness as cellular imaging probes.