Heregulin targets gamma-catenin to the nucleolus by a mechanism dependent on the DF3/MUC1 oncoprotein

The DF3/MUC1 transmembrane oncoprotein is aberrantly overexpressed in most human breast carcinomas and interacts with the Wnt effector gamma-catenin. Here, we demonstrate that MUC1 associates constitutively with ErbB2 in human breast cancer cells and that treatment with heregulin/neuregulin-1 (HRG) increases the formation of MUC1-ErbB2 complexes. The importance of the MUC1-ErbB2 interaction is supported by the demonstration that HRG induces binding of MUC1 and gamma-catenin and targeting of the MUC1-gamma-catenin complex to the nucleolus. Significantly, nucleolar localization of gamma-catenin in response to HRG is dependent on MUC1 expression. Moreover, mutation of a RRK motif in the MUC1 cytoplasmic domain abrogates HRG-induced nucleolar localization of MUC1 and gamma-catenin. In concert with these results, we show nucleolar localization of MUC1 and gamma-catenin in human breast carcinomas but not in normal mammary ductal epithelium. These findings demonstrate that MUC1 functions in cross talk between ErbB2 and Wnt pathways by acting as a shuttle for HRG-induced nucleolar targeting of gamma-catenin.     

Nitric oxide signaling in plant-pathogen interactions

Nitric oxide (NO), first characterized as an endothelium-derived relaxation factor, is involved in diverse cellular processes including neuronal signaling, blood pressure homeostasis, and immune response. Recent studies have also revealed a role for NO as a signaling molecule in plants. As a developmental regulator, NO promotes germination, leaf extension and root growth, and delays leaf senescence and fruit maturation. Moreover, NO acts as a key signal in plant resistance to incompatible pathogens by triggering resistance-associated hypersensitive cell death. In addition, NO activates the expression of several defense genes (e.g. pathogenesis-related genes, phenylalanine ammonialyase, chalcone synthase) and could play a role in pathways leading to systemic acquired resistance.     

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.     

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.     

Fine-scale genetic structure suggests low levels of short-range gene flow in a wolf population of the Italian Apennines

We investigated local gene flow in a high-density wolf (Canis lupus) population of the Italian Apennines, where no effective barrier to wolf dispersal was present. From 1998 to 2004 we examined wolf carcasses and non-invasively collected samples, focusing on three mountain districts, separated by two valleys, where wolf packs showed high spatial stability. Using nine autosomal microsatellites we successfully genotyped 177 samples, achieving the identification of 74 wolves. Genetic relatedness steeply decreased with increasing distance between sampling areas, thus suggesting that short-distance interpack migration is infrequent in this population. In addition, no individual from a central pack under intensive monitoring was sampled in the range of the surrounding packs over a 4-year period. The limited short-distance gene flow resulted in a cryptic genetic structure, which was revealed by Bayesian analysis. A different genetic cluster was found in each of the three mountain areas, and a small proportion of first-generation immigrants was detected. Overall, the present study suggests that local genetic differentiation in Italian wolves might arise from high spatial stability of packs and can be favoured by a combination of long-range dispersal, the attitude to mate between unrelated individuals and a high young mortality rate.     

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.

Objective

We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Results

We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.

Conclusions

MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

Chemopreventive and Antioxidant Activity of the Chamazulene‐Rich Essential Oil Obtained from Artemisia arborescens L. Growing on the Isle of La Maddalena,Sardinia, Italy

The essential oils of Artemisia arborescens growing in Sardinia (Italy), collected during three plant growth stages, i.e., from the vegetative stage to post‐blooming time, were characterized. Moreover, the in vitro antiproliferative and antioxidant activities of the oil isolated from aerial parts collected in February were evaluated. The essential oils belonged to the β‐thujone/chamazulene chemotype, notably with the highest amount of chamazulene (ca. 52%) ever detected up to now in the genus Artemisia and, in general, in essential oils. Quantitative variations in the oil composition were observed as the plant passes from the vegetative to the blooming stage. The oil was tested for its potential tumor cell growth‐inhibitory effect on T98G, MDA‐MB 435S, A375, and HCT116 human cell lines, using the MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) assay. The highest activity was observed on A375 and HCT116 cell lines, with IC₅₀ values of 14 μg/ml. Moreover, the in vitro antioxidant and free radical‐scavenging assays revealed the oil to be an effective scavenger of the ABTS radical cation, with an activity comparable to that of Trolox^®. These results support the use of A. arborescens oil for the treatment of inflamed skin conditions. Finally, the composition of the polar fraction of the A. arborescens aerial parts was also examined, and the main component detected was 5‐O‐caffeoylquinic acid, which was identified for the first time in this plant.