Expression and Functional Studies of Ubiquitin C-Terminal Hydrolase L1 Regulated Genes

Deubiquitinating enzymes (DUBs) have been increasingly implicated in regulation of cellular processes, but a functional role for Ubiquitin C-terminal Hydrolases (UCHs), which has been largely relegated to processing of small ubiquitinated peptides, remains unexplored. One member of the UCH family, UCH L1, is expressed in a number of malignancies suggesting that this DUB might be involved in oncogenic processes, and increased expression and activity of UCH L1 have been detected in EBV-immortalized cell lines. Here we present an analysis of genes regulated by UCH L1 shown by microarray profiles obtained from cells in which expression of the gene was inhibited by RNAi. Microarray data were verified with subsequent real-time PCR analysis. We found that inhibition of UCH L1 activates genes that control apoptosis, cell cycle arrest and at the same time suppresses expression of genes involved in proliferation and migration pathways. These findings are complemented by biological assays for apoptosis, cell cycle progression and migration that support the data obtained from microarray analysis, and suggest that the multi-functional molecule UCH L1 plays a role in regulating principal pathways involved in oncogenesis

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Alterations in gene expression profiles correlated with cisplatin cytotoxicity in the glioma U343 cell line

Gliomas are the most common tumors in the central nervous system, the average survival time of patients with glioblastoma multiforme being about 1 year from diagnosis, in spite of harsh therapy. Aiming to study the transcriptional profiles displayed by glioma cells undergoing cisplatin treatment, gene expression analysis was performed by the cDNA microarray method. Cell survival and apoptosis induction following treatment were also evaluated. Drug concentrations of 12.5 to 300 μM caused a pronounced reduction in cell survival rates five days after treatment, whereas concentrations higher than 25 μM were effective in reducing the survival rates to ~1%. However, the maximum apoptosis frequency was 20.4% for 25 μM cisplatin in cells analyzed at 72 h, indicating that apoptosis is not the only kind of cell death induced by cisplatin. An analysis of gene expression revealed 67 significantly (FDR < 0.05) modulated genes: 29 of which down- and 38 up-regulated. These genes belong to several classes (metabolism, protein localization, cell proliferation, apoptosis, adhesion, stress response, cell cycle and DNA repair) that may represent several affected cell processes under the influence of cisplatin treatment. The expression pattern of three genes (RHOA, LIMK2 and TIMP2) was confirmed by the real time PCR method

Genomics and proteomics approaches to the study of cancer-stroma interactions

<p>Abstract</p> <p>Background</p> <p>The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.</p> <p>Methods</p> <p>The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.</p> <p>Results</p> <p>We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (<it>ARID4A</it>, <it>CALR</it>, <it>GNB2L1</it>, <it>RNF10</it>, <it>SQSTM1</it>, <it>USP9X</it>) were validated by real time PCR.</p> <p>Conclusions</p> <p>A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.</p

Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required

Synthetic versus natural receptors: supramolecular control of chemical sensing in fish

The encapsulation of odorants by the synthetic receptor cucurbit[7]uril (CB[7]) reduces the response of olfactory receptors in Mozambique tilapia (Oreochromis mossambicus) in vivo. For example, the olfactory receptor response to the odorant adamantan-1-amine, as measured by electro-olfactography, was suppressed by 92% in the presence of CB[7]. A reduction in olfactory response of 88% was observed for pentane-1,5-diamine (cadaverine), an odorant associated with carrion avoidance in some fish. The results reveal how the association constants and the concentrations of natural and synthetic receptors play a determinant role and show that synthetic receptors can be used to remove bioactive molecules from fish olfaction

The de-ubiquitinase UCH-L1 is an oncogene that drives the development of lymphoma in vivo by deregulating PHLPP1 and Akt signaling.

Item does not contain fulltextDe-ubiquitinating enzymes (DUBs) can reverse the modifications catalyzed by ubiquitin ligases and as such are believed to be important regulators of a variety of cellular processes. Several members of this protein family have been associated with human cancers; however, there is little evidence for a direct link between deregulated de-ubiquitination and neoplastic transformation. Ubiquitin C-terminal hydrolase (UCH)-L1 is a DUB of unknown function that is overexpressed in several human cancers, but whether it has oncogenic properties has not been established. To address this issue, we generated mice that overexpress UCH-L1 under the control of a ubiquitous promoter. Here, we show that UCH-L1 transgenic mice are prone to malignancy, primarily lymphomas and lung tumors. Furthermore, UCH-L1 overexpression strongly accelerated lymphomagenesis in Emu-myc transgenic mice. Aberrantly expressed UCH-L1 boosts signaling through the Akt pathway by downregulating the antagonistic phosphatase PHLPP1, an event that requires its de-ubiquitinase activity. These data provide the first in vivo evidence for DUB-driven oncogenesis and suggest that UCH-L1 hyperactivity deregulates normal Akt signaling.1 september 201