Glutamate antagonists limit the growth of human cancers in vitro.The mechanism of anticancer action of NMDA antagonists is not known, however. In this article, we report that the NMDA antagonist dizocilpine inhibits the extracellular signal-regulated kinase
Journal of PhysiologyJurkat cells (Benichou et al. 1989;Gulbins et al. 1996), but sparingly expressed in T lymphocytes which lack this capability (van Reyk et al. 2001). We show here that the levels of H + channel expression in human B lymphocytes and in Jurkat cells are quantitatively more than adequate to sustain NADPH oxidase activity in these cells. METHODS CellsJurkat T cells. The human T leukaemia cell line Jurkat E6-1 was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Jurkat T cells were grown in RPMI 1640 medium (GibcoBRL, Eggenstein, Germany) supplemented with 10 % fetal bovine serum and 2 mM glutamine at densities of 1-9 w 10 5 in a 37°C humidified incubator with 5 % CO 2 , and split three times weekly.Human lymphocytes. Venous blood was drawn from healthy adult volunteers after informed written consent was obtained according to procedures approved by the local Institutional Review Board and in accordance with Federal regulations. Lymphocytes were obtained from the whole blood of the donors. The suspension was layered on top of ficoll-hypaque (AmershamPharmarcia, Freiburg, Germany) after dilution with an equal volume of RPMI 1640 (GibcoBRL, Eggenstein, Germany). After centrifugation (30 min, 500 g, room temperature), mononuclear cells were harvested from the interphase, washed twice with lowspeed centrifugation (200 g, 10 min, 20°C) to remove platelets, and resuspended at 1.5 w 10 8 ml _1 in degassed RPMI1640 containing 0.5 % bovine serum albumin and 2 mM EDTA. B cells were separated by negative depletion using the MACS B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. Briefly, after blocking Fc receptors with aggregated human immunoglobulin, cells were incubated with murine hapten-conjugated monoclonal antibodies directed against human CD2, CD4, CD11b, CD16, CD36 and anti-IgE at 10°C for 10 min, washed twice, and then incubated with anti-hapten microbeads at 10°C for 15 min. The washed cell suspension was then placed on a LS+/VS+ column (Miltenyi), and the unlabelled cells passing through were collected from the effluent. This fraction consisted of 97 % CD19+ cells, as judged by flow cytometry after staining with fluoresceinisothiocyanate-conjugated Leu-12 monoclonal antibody (Becton Dickinson, Heidelberg, Germany). Subsequently, the LS+/VS+ column was placed outside the magnetic field, and the retained cells were eluted. This B cell-depleted fraction contained 80-90 % of CD3+ cells as judged by flow cytometry after staining with fluoresceinisothiocyanate-conjugated anti-CD3 monoclonal antibody (Dako, Hamburg, Germany).
We demonstrate that FLX inhibits phosphorylation of ERK1/2 kinases in a time and concentration-dependent manner, followed by reduced phosphorylation of transcription factor c-Myc in A549 and HT29 cells. After treatment with FLX, A549 and HT29 cells demonstrated concentration-dependent decrease in the expression of c-fos, c-jun, cyclin A, cyclin D1, and increased expression of p21 waf1 and p53 genes, which resulted in slowing of the cell cycle progression. We suggest that these changes could be responsible for observed inhibition of cancer cell proliferation during FLX treatment in vitro.
Antagonists at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors limit growth of human cancers in vitro. However, the mechanism of anticancer action of AMPA antagonists is not known. Here we report that the AMPA antagonists GYKI 52466 and CFM-2 inhibit the extracellular signal regulated kinase (ERK1/2) pathway, an intracellular signaling cascade which is activated by growth factors and controls proliferation of lung adenocarcinoma cells. AMPA antagonists reduced phosphorylation of cAMP-responsive element binding protein (CREB), suppressed expression of cyclin D1, upregulated the cell cycle regulators and tumor suppressor proteins p21 and p53 and decreased number of lung adenocarcinoma cells in G2 and S phases of the cell cycle. These findings reveal potential mechanism of antiproliferative action of AMPA antagonists and indicate that this class of compounds may be useful in the therapy of human cancers.
ABSTRACT:The ductus arteriosus (DA), a fetal arterial shunt vessel between the proximal descending aorta and the pulmonary artery, closes shortly after birth. Initial functional closure as a result of the DA's smooth muscle contraction is followed by definite anatomical closure. The latter involves several complex mechanisms like endothelial cushion formation and smooth muscle cell migration resulting in fibrosis and sealing of the vessel. These complex steps indicate highly specialized functions of the DA vascular smooth muscle cells (VSMCs), endothelial cells, and fibroblasts. Herein, we describe a new reproducible method for isolating VSMCs, endothelial cells, and fibroblasts of high viability from fetal rat DA using immunomagnetic cell sorting. Purity of the different cell cultures was assessed by immunohistochemistry and flow cytometry and ranged between 85 and 94%. The capability of the VSMCs to react to hypoxic stimuli was assessed by intracellular calcium and ATP measurements and by VEGF mRNA expression analysis. VSMCs respond to hypoxia with decreases in intracellular calcium concentrations and ATP levels, whereas VEGF mRNA expression increased 3.2-fold. The purified vessel-specific different cell types are suitable for subsequent gene expression profiling and functional studies and provide important tools for improving our understanding of the complex processes involved in the closure of the DA. T he ductus arteriosus (DA) is a shunt which connects the proximal descending aorta and the main pulmonary artery (PA). During fetal life, it serves to bypass the pulmonary circulation. The DA closes shortly after birth, and pulmonary blood flow increases during late gestation (1). After birth, an abrupt increase in oxygen tension results in ductal constriction, which is further promoted by falling levels of prostaglandin E2 because of an increased pulmonary metabolism and the elimination of placental prostaglandin. DA medial smooth muscle contraction leads to wall thickening, luminal obliteration, and shortening of the vessel. Permanent sealing of the DA is a process generated by the infolding of the endothelium, neointima formation, subintimal disruption, and vasa vasorum ingrowth resulting in subsequent vessel fibrosis. Several major molecular pathways are known to be involved in this programmed proliferation during permanent closure of the DA, e.g. NO signaling, the system of VEGF-promoted angiogenesis, and the cyclooxygenase/prostaglandin system (2-5).However, many questions concerning the exact function and cross-talk of these signaling pathways and the molecular basis for failed DA closure still remain unanswered (6). Previous studies investigating molecular events in the duct were in part hampered by the lack of an in vitro model that allows to study the processes in question separately in each of the different cell types of the DA instead of using the whole vessel (7). Herein, we describe an improved and reproducible method to culture organotypic vascular smooth muscle cells (VSMCs), fibroblasts, and e...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.