Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin

The glycoprotein hormone, human chorionic gonadotropin (hCG), contains both N- and O-linked oligosaccharide chains linked to its beta-subunit. Using the human choriocarcinoma cell line, BeWo, we have examined the temporal relationship between N- and O-glycosylation of hCG and the subsequent processing of both types of oligosaccharide chains. The results indicate that, as observed in related cell lines, mature, completely glycosylated forms of the subunits of hCG cannot be detected intracellularly in BeWo cells during pulse-chase experiments with [35S]methionine. To more directly study the temporal relationship between N- and O-glycosylation of hCG in BeWo cells, 14C-amino acids and [3H]glucosamine (which also serves as a precursor to N-acetylgalactosamine) were used to label hCG. The results of these studies are consistent with a model for the N- and O-glycosylation of hCG in which 1) N-glycosylation of hCG occurs co-translationally or very shortly after translation, and 2) the addition of O-linked GalNAc residues to the polypeptide and the addition of peripheral GlcNAc residues to the N-linked oligosaccharide chains occur just prior to secretion, presumably in the Golgi complex.     

Side population cells in anaplastic thyroid cancer and normal thyroid

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated.One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12.This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.     

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.     

Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue

Over-expression of hexose transporters (Gluts), specifically Glut-1, is a common event in human malignancies. In prostate cancer (CaP), however, expression of Gluts has been characterized poorly. In this study, expression and distribution of Glut-1 and Glut-5 proteins were characterized using immunohistochemistry in 76 specimens of benign prostate, 10 specimens of high-grade intraepithelial neoplasia (HGPIN), and 28 specimens of CaP. In addition, mRNA expression of Glut-2, Glut-7, Glut-9, and Glut-11 was analyzed in a set of five specimens of benign prostate and CaP. In benign prostate, Glut-1 localized to the basal cells and to the basolateral membrane of secretory/luminal epithelial cells. Glut-5, however, localized to the apical membrane of secretory/luminal epithelial cells. In HGPIN, Glut-1 was immunohistochemically undetectable. Glut-5, however, localized to the apical membrane of the neoplastic epithelial cells. In CaP, Glut-1 and Glut-5, were immunohistochemically undetectable. However, over-expression of GLUT1 was observed in some specimens of highly proliferative intraductal CaP. Glut-7, Glut-9, and Glut-11 mRNAs were detected in benign prostate and CaP, however, only Glut-11 mRNA was consistently up-regulated in CaP compared to benign prostate. Low levels of expression of Glut-1 protein in the majority of CaP could explain, at least in part, the limited clinical applicability of positron emission tomography using 2-[18F]-fluoro-2-deoxy-D-glucose for imaging CaP. Moreover, expression of Glut-5 in HGPIN suggested that fructose could be utilized as potential metabolic substrate in HGPIN. Understanding the molecular mechanisms involved in regulation/dysregulation of Gluts in CaP could provide insight in the understanding of hexose metabolism in CaP.     

Antithyroid cancer effects of human neural stem cells expressing therapeutic genes on anaplastic thyroid cancer cells

Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-β (IFN-β) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-β suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-β cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-β cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-β. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.     

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.     

Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.     

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.     

增龄对大鼠膈肌Glut-1和Glut-4 mRNA表达的影响

目的研究大鼠膈肌Glut-1和Glut-4 mRNA的增龄变化情况,进一步探讨增龄对呼吸肌代谢及功能变化的机制。方法雄性健康清洁级SD大鼠24月龄7只、3月龄12只,应用RT-PCR方法检测大鼠膈肌Glut-1、Glut-4 mRNA表达。结果老年组大鼠Glut-1、Glut-4 mRNA表达较年轻组表达明显升高。结论大鼠膈肌不同于单纯骨骼肌和心肌,Glut-1、Glut-4 mRNA的表达具有特殊的增龄趋势。     

Expression of Glut-4 and Glut-1 transporters in rat diaphragm muscle

Glucose transporters (Gluts) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. Generally, alterations of Gluts expression in limb skeletal muscle have been reported. However, the changes of Glut isoforms in respiratory muscle which contracts with a duty cycle have rarely been studied. This study was performed to evaluate at the light microscopy level the expression of Glut-4 and Glut-1 transporters in normal and denervated diaphragm by immunohistochemistry method with specific Gluts antibodies. The results showed Glut-4 immunoreactivity in both the cell periphery and the interior of myocytes. Glut-1 was also present in the cell border and in the interior of myocytes in control diaphragm. However, Glut-4 staining was stronger than Glut-1 staining in control diaphragm. In denervated hemidiaphragm, the Glut-4 immunolabelling decreased and Glut-1 increased. These data indicated that (1) Glut-4 and Glut-1 transporters were observed in diaphragm; and (2) there were alterations in the expression of both glucose transporters after denervation. These alterations in Glut isoforms after denervation may be associated with the removal of innervation itself, and/or may partly result from passive stretch imposed by inspiratory activation of the contralateral side.     

The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.     

RNA-Seq transcriptome analysis reveals Maackia amurensis leukoagglutinin has antitumor activity in human anaplastic thyroid cancer cells

Molecular Biology Reports - Lectins are carbohydrate-binding molecules that can bind specifically to the sugar residues of glycoconjugates and are found in almost all organisms. Plant lectins...     

Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells

Glycosylation is one of the most common modifications of proteins and lipids and also a major source of biological diversity in eukaryotes. It is critical for many basic cellular functions and recognition events that range from protein folding to cell signaling, immunological defense, and the development of multicellular organisms. Glycosylation takes place mainly in the endoplasmic reticulum and Golgi apparatus and involves dozens of functionally distinct glycosidases and glycosyltransferases. How the functions of these enzymes, which act sequentially and often competitively, are coordinated to faithfully synthesize a vast array of different glycan structures is currently unclear. Here, we investigate the supramolecular organization of the Golgi N- and O-glycosylation pathways in live cells using a FRET flow cytometric quantification approach. We show that the enzymes form enzymatically active homo- and/or heteromeric complexes within each pathway. However, no complexes composed of enzymes that operate in different pathways, were detected, which suggests that the pathways are physically distinct. In addition, we show that complex formation is mediated almost exclusively by the catalytic domains of the interacting enzymes. Our data also suggest that the heteromeric complexes are functionally more important than enzyme homomers. Heteromeric complex formation was found to be dependent on Golgi acidity, markedly impaired in acidification-defective cancer cells, and required for the efficient synthesis of cell surface glycans. Collectively, the results emphasize that the Golgi glycosylation pathways are functionally organized into complexes that are important for glycan synthesis.     

Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload

Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca²⁺) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca²⁺ modulation is expected to be a novel therapeutic target in cancers. However, whether Ca²⁺ modulation is effective in ATC therapy remains unknown. In this study, we reported that capsaicin (CAP), a transient receptor potential vanilloid type1 (TRPV1) agonist, inhibited the viability of anaplastic thyroid cancer cells. Capsaicin treatment triggered Ca²⁺ influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca²⁺ concentration was mirrored in the mitochondria and caused a severe condition of mitochondrial calcium overload in ATC cells. In addition, the disruption of mitochondrial calcium homeostasis caused by capsaicin led to mitochondrial dysfunction in ATC cells, as evidenced by the production of mitochondrial reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), and opening of mitochondrial permeability transition pore (mPTP). Next, the resulting release of cyt c into the cytosol triggered apoptosome assembly and subsequent caspase activation and apoptosis. It was worth noting that both TRPV1 antagonist (capsazepine) and calcium chelator (BAPTA) could attenuate aberrant Ca²⁺ homeostasis, mitochondrial dysfunction and apoptosis induced by capsaicin treatment. Thus, our study demonstrated that capsaicin induced mitochondrial calcium overload and apoptosis in ATC cells through a TRPV1-mediated pathway. The better understanding of the anti-cancer mechanisms of calcium modulation provides a potential target for the ATC therapy.