Cyclin D1 is a direct target of JAG1-mediated Notch signaling in breast cancer.

The Notch ligand, JAG1 is associated with breast cancer recurrence. Herein, we report on a genomics approach to elucidate mechanisms downstream of JAG1 that promote breast cancer growth. In a survey of 46 breast cancer cell lines, we found that triple negative (TN; basal and mesenchymal ER-, PR-, and Her2-negative) lines express JAG1 at significantly higher levels than do HER2(+) or luminal (ER(+)) Her2(-) cell lines. In contrast to the luminal lines tested (T47D and MCF7), TN breast cancer cell lines (HCC1143 and MDA MB231) display high-level JAG1 expression and growth inhibition with RNA interference-induced JAG1 down-regulation. We used microarray profiling of TN tumor cells transfected with JAG1 siRNA to identify JAG1-regulated genes (P or=1.5). Among JAG1-regulated genes identified, cyclin D1 was found to be a direct target of NOTCH1 and NOTCH3. We show that JAG1 down-regulation reduces direct binding of Notch to the cyclin D1 promoter, reduced cyclin D1 expression and inhibition of cell cycle progression through the cyclin D1-dependant G1/S checkpoint. Furthermore, we show that cyclin D1 and JAG1 expression correlate in TN breast cancer expression datasets. These data suggest a model whereby JAG1 promotes cyclin D1-mediated proliferation of TN breast cancers.

Design and analysis of quantitative differential proteomics investigations using LC-MS technology.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is becoming an increasingly important tool in characterizing the abundance of proteins in biological samples of various types and across conditions. Effects of disease or drug treatments on protein abundance are of particular interest for the characterization of biological processes and the identification of biomarkers. Although state-of-the-art instrumentation is available to make high-quality measurements and commercially available software is available to process the data, the complexity of the technology and data presents challenges for bioinformaticians and statisticians. Here, we describe a pipeline for the analysis of quantitative LC-MS data. Key components of this pipeline include experimental design (sample pooling, blocking, and randomization) as well as deconvolution and alignment of mass chromatograms to generate a matrix of molecular abundance profiles. An important challenge in LC-MS-based quantitation is to be able to accurately identify and assign abundance measurements to members of protein families. To address this issue, we implement a novel statistical method for inferring the relative abundance of related members of protein families from tryptic peptide intensities. This pipeline has been used to analyze quantitative LC-MS data from multiple biomarker discovery projects. We illustrate our pipeline here with examples from two of these studies, and show that the pipeline constitutes a complete workable framework for LC-MS-based differential quantitation. Supplementary material is available at http://iec01.mie.utoronto.ca/~thodoros/Bukhman/.

Synthesis and characterization of a hemoglobin-ribavirin conjugate for targeted drug delivery.

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.

Targeted delivery of ribavirin improves outcome of murine viral fulminant hepatitis via enhanced anti-viral activity.

Side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in hepatitis C virus (HCV) patients. Coupling ribavirin to macromolecular carriers that target the drug to the liver would reduce systemic complications. The aim of this study was to evaluate the efficacy of a hemoglobin-ribavirin conjugate (HRC 203) in murine hepatitis virus strain 3 (MHV-3) induced viral hepatitis. HRC 203 had greater anti-viral activity on both isolated hepatocytes and macrophages, whereas both ribavirin and HRC 203 inhibited production of the pro-inflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) by macrophages. In vivo, untreated MHV-3-infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day 4 post infection. Livers recovered from untreated infected mice showed greater than 90% necrosis. In contrast, survival was enhanced in both ribavirin- and HRC 203-treated mice with a marked reduction in biochemical [ALT(max) 964 +/- 128 IU/L (ribavirin); 848 +/- 212 IU/L (HRC 203)] and histological evidence of hepatic necrosis (<10% in ribavirin/HRC 203 vs. 90% in untreated controls). Clinically, HRC 203-treated mice behaved normally, in contrast to ribavirin-treated mice, which developed lethargy and abnormal fur texture. In conclusion, targeted delivery of ribavirin to the liver alters the course of MHV-3 infection as demonstrated by prolonged survival, improved behavior, and reduced signs of histologically evident disease, as well as inhibition of viral replication and production of inflammatory cytokines in vitro.