Alessandra Sayuri Kikuchi Tamajusuku

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.

Gastrin-Releasing Peptide Receptors Regulate Proliferation of C6 Glioma Cells through a Phosphatidylinositol 3-Kinase-Dependent Mechanism

Gastrin-releasing peptide (GRP) has been proposed as a major growth factor in brain tumors, and GRP receptor (GRPR) antagonists show antiproliferative effects in experimental gliomas. However, the underlying molecular events downstream of GRPR activation remain poorly understood. In the present study, we examined the role of the GRPR in regulating proliferation of glioma cells in vitro and its possible interaction with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Expression of GRPR mRNA and protein in C6, U-87MG, and U-373MG glioma cells was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Proliferation of C6 and U-87MG, but not U-373MG cells was significantly inhibited by the GRPR antagonist RC-3095, whereas the GRPR agonist bombesin (BB) significantly enhanced proliferation of C6 cells. The BB-induced stimulatory effect on cell proliferation was prevented by either RC-3095 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Our results provide the first evidence that the GRPR regulates proliferation of C6 glioma cells and suggest that PI3K is required for GRPR-mediated stimulation of glioma growth.

Characterization of ATP-Induced Cell Death in the GL261 Mouse Glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010.

Activity and expression of ecto-5′-nucleotidase/CD73 are increased by thyroid hormones in vascular smooth muscle cells

Extracellular nucleotides ATP, ADP, AMP and adenosine are well known signaling molecules of the cardiovascular system that are involved in several physiological processes: cell proliferation, platelet aggregation, inflammatory processes and vascular tonus. The levels of these molecules are controlled by ecto-NTPDases and ecto-5′-nucleotidase/CD73 (ecto-5′-NT/CD73) actions, which are responsible for the complete ATP degradation to adenosine. The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), play important roles in the vascular system promoting vasodilatation. Here we investigated the influence of thyroid hormones on the enzyme cascade that catalyzes the interconversion of purine nucleotides in vascular smooth muscle cells (VSMC). Exposure of VSMCs to 50nM T3 or T4 did not change ATP and ADP hydrolysis significantly. However, the same treatment caused an increase of 75% in AMP hydrolysis, which was time-dependent but dose-independent. Moreover, T3 treatment significantly increased ecto-5′-NT/CD73 mRNA expression, which suggests a genomic effect of this hormone upon ecto-5′-NT/CD73. In addition to the importance of the ecto-5′-NT in cell proliferation and differentiation, its overexpression could result in higher extracellular levels of adenosine, an important local vasodilatator molecule.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference.

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down‐regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild‐type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial‐specific GFAP, while the presence of beta‐3 tubulin‐positive cells diminished at the same time. Proliferation induction in the presence of stable anti‐P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down‐regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models.

P2X7 receptor as predictor gene for glioma radiosensitivity and median survival

Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival.

Óleo de cravo como anestésico para guppy

The present study evaluated different concentrations of clove oil for anesthesia of guppy (Poecilia reticulata). Adult female, male and juveniles fish were used to assess the influence of the anesthesia. The animals were individually submitted to the following clove oil concentrations: 50, 75, 100, 125 and 150 mg L-1. The induction, recovery stages and mortality 96 hours after the experiment were evaluated. All fish exposed to experimental concentrations reached the final stage of anesthesia. Adult female and male and juveniles guppies respond differently to anesthesia induction by clove oil. Thus, it is recommended the use of clove oil on concentrations of 125 mg L-1 for adult males and from 75 mg L-1 to 150 mg L-1 for adult females and juveniles animals.

Analysis of NTPDase2 in the cell membrane using fluorescence recovery after photobleaching (FRAP)

NTPDase2, a member of the CD39/NTPDase family, is an ecto‐nucleotidase anchored to the plasma membrane by two transmembrane domains, with a catalytic site facing the extracellular space and preferentially hydrolyzing nucleoside triphosphates. While NTPDase2 is expressed in many cell types, its unique functionality, mobility and dynamics at the cell membrane remain unexplored. We therefore constructed a recombinant NTPDase2 linked to the yellow fluorescent protein (EYFP) to investigate its dynamics by confocal microscopy. The present study shows that the expression of EYFP‐NTPDase2 in different cell lines does not affect its proliferation, migration and adhesion to extracellular matrices (ECM). Moreover, in human embryonic kidney cells 293 (HEK293) grown on collagen type I and fibronectin, EYFP‐NTPDase2 fluorescence is greater in free plasma membrane regions than in cell–cell contacts, in comparison with cells grown on other substrates. Differences in the time required for fluorescence recovery after photobleaching (FRAP) in free membrane regions and cell–cell contacts indicate that the mobility of EYFP‐NTPDase2 depends on the matrix to which the cells are attached.

Fillet and carcass yield and fillet chemical composition of piava from fish farming and from the wild

Piava (Leporinus obtusidens) is one of the main cultivated native fish and one of the most caught in Rio Grande do Sul, South Brazil. The objective of this work was to compare the carcass and fillet yields of piava, females and males, collected from the wild (Treatment 1 T1) and from fish culture (Treatment 2 T2) and to analyze the chemical composition of fillets. For this purpose, four females and four males of each treatment were used. T1 females had significant higher values (P<0.05) in the carcass yield. Chemical analyses of the fillet indicated that fish originating from fish farming, independent of sex, showed higher values (P<0.05) for protein and ash contents. Thus, the management and the suitable food in fish farming could contribute to the production of a better quality fish.

Abstract A195: The role of P2X7 receptor in glioma cells.

P2X7 is an ionotropic receptor, whose activation by extracellular ATP can cause pro- or anti-apoptotic responses, depending on the concentration of the ligand. This receptor is expressed in almost all the cell types of the central nervous system and has been linked to cell death in several cells. Gliomas are primary tumors that arise in the CNS, and glioblastoma is the most deadly brain tumor due to their high proliferation, migration and infiltrative nature. Here, we show that survival of patients with gliomas directly correlate with the expression of P2X7 purinergic receptor analyzed with immunohistochemistry of a local group of patients or microarray in a set of patients from “REMBRANDT” and “The Cancer Genome Atlas” databases. A sub-population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP-induced cell death, decreased cell adhesion and promoted migration, suggesting that this receptor is necessary for this effect. Since these are features of more aggressive cancers, this could be part of the explanation for the correlation observed in the patients’ outcome. These data support the importance of the purinergic system in general and the P2X7 receptor particularly in the pathobiology of gliomas. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A195. Citation Format: Alessandra S. K Tamajusuku, Franciele C. Kipper, Eduardo C. Filippi-Chiela, Debora G. Flores, Gleice M. Reder, Luise Meurer, Ana M.O. Battastini, Rafael Roesler, Guido Lenz, Marcia R. Wink. The role of P2X7 receptor in glioma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A195.