scholarly journals Proteoglycan involvement in polyamine uptake

1999 ◽  
Vol 338 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Mattias BELTING ◽  
Susanne PERSSON ◽  
Lars-Åke FRANSSON
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Selective Inhibition ◽  
Lung Fibroblasts ◽  
Ovary Cell ◽  
Wild Type ◽  
Polyamine Biosynthesis ◽  
Heparan Sulphate Proteoglycans ◽  
Mutant Cells

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki = 0.16±0.04 µM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-β-d-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with α-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by α-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or β-d-xyloside, which might have future therapeutical implications.

scholarly journals Sulphated and undersulphated heparan sulphate proteoglycans in a Chinese hamster ovary cell mutant defective in N-sulphotransferase

1994 ◽  
Vol 303 (1) ◽  
pp. 81-87 ◽  
Author(s):  
K J Bame ◽  
L Zhang ◽  
G David ◽  
J D Esko
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Cell Mutant ◽  
Heparan Sulphate Proteoglycans ◽  
Chain Polymerization

The Chinese hamster ovary cell mutant, pgsE-606, synthesizes undersulphated heparan sulphate glycosaminoglycans because of a deficiency in N-sulphotransferase activity [Bame and Esko (1989) J. Biol. Chem. 264, 8059-8065]. We compared the heparan sulphate proteoglycans synthesized by mutant and wild-type cells to determine what effect the undersulphation defect had on proteoglycan structure. The majority of heparan sulphate proteoglycans synthesized by pgsE-606 were undersulphated, but the mutant also synthesized a population of proteoglycans that were sulphated to the same extent as wild-type molecules. Anion-exchange analysis of the glycosaminoglycans in each proteoglycan population showed that they were all modified in the same way. The length of the glycosaminoglycans in each proteoglycan population were similar, suggesting that N-sulphation does not affect chain polymerization. To examine whether the sulphation state of the attached heparan sulphate glycosaminoglycans was dependent on the protein core, we purified syndecan-1 from mutant and wild-type cells using antibodies against the core protein. As with the unfractionated heparan sulphate proteoglycans, pgsE-606 synthesized both undersulphated and sulphated syndecan-1. Each pool contained either undersulphated or sulphated glycosaminoglycan chains respectively. Thus the modification of all heparan sulphate chains on a core protein occurs on a proteoglycan-wide basis (i.e. to the same extent).

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Fatty Acid Remodeling of GPI-anchored Proteins Is Required for Their Raft Association

2007 ◽  
Vol 18 (4) ◽  
pp. 1497-1506 ◽  
Author(s):  
Yusuke Maeda ◽  
Yuko Tashima ◽  
Toshiaki Houjou ◽  
Morihisa Fujita ◽  
Takehiko Yoko-o ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Rafts ◽  
Cho Cells ◽  
Membrane Fraction ◽  
Double Mutant ◽  
Chinese Hamster ◽  
Wild Type ◽  
Saturated Chain ◽  
Mutant Cells ◽  
Detergent Resistant Membrane

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3. The surface GPI-APs isolated from the PGAP2 and -3 double-mutant Chinese hamster ovary (CHO) cells had unsaturated chains, such as oleic, arachidonic, and docosatetraenoic acids in the sn-2 position, whereas those from wild-type CHO cells had exclusively stearic acid, a saturated chain, indicating that the sn-2 chain is exchanged to a saturated chain. We then assessed the association of GPI-APs with lipid rafts. Recovery of unremodeled GPI-APs from the double-mutant cells in the detergent-resistant membrane fraction was very low, indicating that GPI-APs become competent to be incorporated into lipid rafts by PGAP3- and PGAP2-mediated fatty acid remodeling. We also show that the remodeling requires the preceding PGAP1-mediated deacylation from inositol of GPI-APs in the endoplasmic reticulum.

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

1989 ◽  
Vol 109 (6) ◽  
pp. 3157-3167 ◽  
Author(s):  
C L Schreiner ◽  
J S Bauer ◽  
Y N Danilov ◽  
S Hussein ◽  
M M Sczekan ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Plasma Membranes ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Fibronectin Receptor ◽  
Alpha Chain ◽  
Isolation And Characterization

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.

Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin

1990 ◽  
Vol 96 (3) ◽  
pp. 519-526
Author(s):  
L.B. Joseph ◽  
D.L. Kreutzer ◽  
M.L. Tanzer
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Spreading ◽  
Immunohistochemical Analysis ◽  
Cell Types ◽  
Wild Type ◽  
Fibronectin Receptor ◽  
Cell Extracts ◽  
Mutant Cells

The development of receptor-defective or -deficient mutants can be applied to the investigation of cell-matrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (P less than 0.001) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (alpha FN/beta 1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the beta 1 subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the alpha and beta 1 subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of bovine viral diarrhoea virus glycoprotein Erns with cell surface glycosaminoglycans

Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 451-459 ◽  
Author(s):  
Munir Iqbal ◽  
Helen Flick-Smith ◽  
John W. McCauley
Keyword(s):  
Cho Cells ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Flow Cytometric Analysis ◽  
Chinese Hamster ◽  
Bovine Viral Diarrhoea Virus ◽  
Bovine Viral Diarrhoea ◽  
High Concentration ◽  
Mammalian Cell Lines ◽  
Diarrhoea Virus

Recombinant Erns glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble Erns to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged Erns to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. Erns failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. Erns also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of Erns binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-l-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of Erns to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction.

scholarly journals Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

1990 ◽  
Vol 110 (2) ◽  
pp. 295-308 ◽  
Author(s):  
K M Cadigan ◽  
D M Spillane ◽  
T Y Chang
Keyword(s):  
Cholesteryl Ester ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Ovary Cell ◽  
Ester Synthesis ◽  
Cholesterol Trafficking ◽  
Isolation And Characterization ◽  
Mutant Cells

This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)-cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

scholarly journals Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

2004 ◽  
Vol 279 (44) ◽  
pp. 46035-46045 ◽  
Author(s):  
Fengyu Qiao ◽  
Jun Mi ◽  
James B. Wilson ◽  
Gang Zhi ◽  
Natalie R. Bucheimer ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Mutant Cells

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others (Qiao, F., Moss, A., and Kupfer, G. M. (2001)J. Biol. Chem. 276, 23391–23396 and Futaki, M., Watanabe, S., Kajigaya, S., and Liu, J. M. (2001)Biochem. Biophys. Res. Commun. 281, 347–351) have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in anin vitrokinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-typeFANCGcDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.

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