Localization of the Epstein-Barr virus protein LMP 1 to exosomes

The Epstein-Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane, Co-labelling of cells with both an LIMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LIMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LIMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation

Abstract - Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. Methodology/Principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. Conclusions/Significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

In vivo and in vitro effects of RAD001 on bladder cancer

Objective: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. Methods: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. Results: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P 0.001), was only observed in the 5637 cell line. Conclusion: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Cellular uptake of BCG-loaded chitosan microparticles and in vivo evaluation of immune response following intranasal immunisation

Attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only currently available vaccine against tuberculosis. It is highly effective in pre-exposure immunisation against TB in children when administered by subcutaneous route to newborns. However, it does not provide permanent protection in adults. In this work, polymeric chitosan-alginate microparticles have been evaluated as potential nasal delivery systems and mucosal adjuvants for live attenuated BCG. Chitosan (CS) has been employed as adjuvant and mucosal permeation-enhancer, and, together with alginate (ALG), as additive to enhance BCG-loaded microparticles (MPs) cellular uptake in a human monocyte cell line, by particle surface modification. The most suitable particles were used for vaccine formulation and evaluation of immune response following intranasal immunisation of BALB/c mice.

Chemical and biological studies from an Azorean macroalga: Ulva rigida

3rd Portuguese Meeting on Medicinal Chemistry and 1st Portuguese-Spanish-Brazilian Meeting on Medicinal Chemistry, Aveiro, 28-30 Novembro 2012.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 μM; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 μM, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Evaluation of 99mTc-Sestamibi as a potential tool to investigate PgP activity in inflammation

Introduction: In the XXI Century ’s Society the scientific investigation process has been growing steadily , and the field of the pharmaceutical research is one of the most enthusiastic and relevant . Here, it is very important to correlate observed functional alterations with possibly modified drug bio distribution patterns . Cancer, inflammation and inf ection are processes that induce many molecular intermediates like cytokines, chemokines and other chemical complexes that can alter the pharmacokinetics of many drugs. One cause of such changes is thought to be the modulator action of these complexes in t he P - Glyco p rotein activity, because they can act like inducers/inhibitors of MDR - 1 expression. This protein results from the expression of MDR - 1 gene, and acts as an ATP energy - dependent efflux pump, with their substrates including many drugs , like antiretrovirals, anticancers, anti - infectives, immunosuppressants, steroids or opioids . Objectives: Because of the lack of methods to provide helpful information in the investigation of in vivo molecular changes in Pgp activity during infection/infl ammation processes, and its value in the explanation of the altered drug pharmacokinetic, this paper want to evaluate the potential utility of 99m Tc - Sestamibi scintigraphy during this kind of health sciences investigation. Although the a im is indeed to create a technique to the in vivo study of Pgp activity, this preliminary Project only reaches the in vitro study phase, assumed as the first step in a n evaluation period for a new tool development. Materials and Methods: For that reason , we are performing in vitro studies of influx and efflux of 99m Tc - Sestamibi ( that is a substrate of Pgp) in hepatocytes cell line (HepG2). We are interested in clarify the cellular behavior of this radiopharmaceutical in Lipopolysaccharide(LPS) stimulated cells ( well known in vitro model of inflammation) to possibly approve this methodology. To validate the results, the Pgp expression will be finally evaluated using Western Blot technique. Results: Up to this moment , we still don’t have the final results, but we have already enough data to let us believe that LPS stimulation induce a downregulation of MDR - 1, and consequently Pgp, which could conduce to a prolonged retention of 99m Tc - Sestamibi in the inflamed cells . Conclusions: If and when this methodology demonstrate the promising results we expect, one will be able to con clude that Nuclear Medicine is an important tool to help evidence based research also on this specific field .

Mutation accumulation in Tetrahymena

Background - The rate and fitness effects of mutations are key in understanding the evolution of every species. Traditionally, these parameters are estimated in mutation accumulation experiments where replicate lines are propagated in conditions that allow mutations to randomly accumulate without the purging effect of natural selection. These experiments have been performed with many model organisms but we still lack empirical estimates of the rate and effects of mutation in the protists. Results - We performed a mutation accumulation (MA) experiment in Tetrahymena thermophila, a species that can reproduce sexually and asexually in nature, and measured both the mean decline and variance increase in fitness of 20 lines. The results obtained with T. thermophila were compared with T. pyriformis that is an obligate asexual species. We show that MA lines of T. thermophila go to extinction at a rate of 1.25 clonal extinctions per bottleneck. In contrast, populations of T. pyriformis show a much higher resistance to extinction. Variation in gene copy number is likely to be a key factor in explaining these results, and indeed we show that T. pyriformis has a higher mean copy number per cell than T. thermophila. From fitness measurements during the MA experiment, we infer a rate of mutation to copy number variation of 0.0333 per haploid MAC genome of T. thermophila and a mean effect against copy number variation of 0.16. A strong effect of population size in the rate of fitness decline was also found, consistent with the increased power of natural selection. Conclusions - The rate of clonal extinction measured for T. thermophila is characteristic of a mutational degradation and suggests that this species must undergo sexual reproduction to avoid the deleterious effects detected in the laboratory experiments. We also suggest that an increase in chromosomal copy number associated with the phenotypic assortment of amitotic divisions can provide an alternative mechanism to escape the deleterious effect of random chromosomal copy number variation in species like T. pyriformis that lack the resetting mechanism of sexual reproduction. Our results are relevant to the understanding of cell line longevity and senescence in ciliates.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

eRF3a/GSPT1 12-GGC allele increases the susceptibility for breast cancer development

It is now widely recognized that translation factors are involved in cancer development and that components of the translation machinery that are deregulated in cancer cells may become targets for cancer therapy. The eukaryotic Release Factor 3 (eRF3) is a GTPase that associates with eRF1 in a complex that mediates translation termination. eRF3a/GSPT1 first exon contains a (GGC)n expansion coding for proteins with different N-terminal extremities. Herein we show that the longer allele (12-GGC) is present in 5.1% (7/137) of the breast cancer patients analysed and is absent in the control population (0/135), corresponding to an increased risk for cancer development, as revealed by Odds Ratio analysis. mRNA quantification suggests that patients with the 12-GGC allele overexpress eRF3a/GSPT1 in tumor tissues relative to the normal adjacent tissues. However, using an in vivo assay for translation termination in HEK293 cells, we do not detect any difference in the activity of the eRF3a proteins encoded by the various eRF3a/GSPT1 alleles. Although the connection between the presence of eRF3a/GSPT1 12-GGC allele and tumorigenesis is still unknown, our data suggest that the presence of the 12-GGC allele provides a potential novel risk marker for various types of cancer.

Modulation of translation factor's gene expression by histone deacetylase inhibitors in breast cancer cells

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

Synthesis, structural characterization and leishmanicidal activity evaluation of ferrocenyl N-heterocyclic compounds

Agência Financiadora: FCT - PTDC/QUI/72656/2006 ; SFRH/BPD/27454/2006; SFRH/BPD/44082/2008; SFRH/BPD/41138/2007

Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to BCG immunotherapy

High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T). METHODS: In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn. RESULTS: From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR=2.668; (1.344-5.254); P=0.005), maintenance schedule (HR=0.480; (0.246-0.936); P=0.031) and multifocality (HR=2.065; (1.033-4.126); P=0.040). sTn or s6T expression was associated with BCG response (P=0.024; P<0.0001) and with increased recurrence-free survival (P=0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR=0.296; (0.148-0.594); P=0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death. CONCLUSION: s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy.

Overexpression of tumour-associated carbohydrate antigen sialyl-Tn in advanced bladder tumours

Little is known on the expression of the tumour-associated carbohydrate antigen sialyl-Tn (STn), in bladder cancer. We report here that 75% of the high-grade bladder tumours, presenting elevated proliferation rates and high risk of recurrence/progression expressed STn. However, it was mainly found in non-proliferative areas of the tumour, namely in cells invading the basal and muscle layers. STn was also found in tumour-adjacent mucosa, which suggests its dependence on a field effect of the tumour. Furthermore, it was not expressed by the normal urothelium, demonstrating the cancer-specific nature of this antigen. STn expression correlated with that of sialyltransferase ST6GalNAc.I, its major biosynthetic enzyme. The stable expression of ST6GalNAc.I in the bladder cancer cell line MCR induced STn expression and a concomitant increase of cell motility and invasive capability. Altogether, these results indicate for the first time a link between STn expression and malignancy in bladder cancer. Hence, therapies targeting STn may constitute new treatment approaches for these tumours.