scholarly journals Genetic Evidence for ATP-dependent Endoplasmic Reticulum-to-Golgi Apparatus Trafficking of Ceramide for Sphingomyelin Synthesis in Chinese Hamster Ovary Cells

1999 ◽  
Vol 144 (4) ◽  
pp. 673-685 ◽  
Author(s):  
Masayoshi Fukasawa ◽  
Masahiro Nishijima ◽  
Kentaro Hanada
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Chinese Hamster Ovary ◽  
De Novo ◽  
Chinese Hamster ◽  
Atp Depletion ◽  
Ovary Cell ◽  
Dependent Manner ◽  
Wild Type ◽  
A Cells

LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse–chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.

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.

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.

scholarly journals A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions.

1984 ◽  
Vol 99 (4) ◽  
pp. 1296-1308 ◽  
Author(s):  
A R Robbins ◽  
C Oliver ◽  
J L Bateman ◽  
S S Krag ◽  
C J Galloway ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Vesicular Stomatitis Virus ◽  
Chinese Hamster Ovary ◽  
Sindbis Virus ◽  
Chinese Hamster ◽  
Proteolytic Processing ◽  
Ovary Cell ◽  
Single Mutation ◽  
Common Component ◽  
Vesicular Stomatitis

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

scholarly journals Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations.

1981 ◽  
Vol 1 (7) ◽  
pp. 652-660 ◽  
Author(s):  
M Meuth
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Genetic Locus ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Single Mutation ◽  
Wild Type ◽  
Ovary Cells ◽  
Mutator Gene ◽  
Mutator Activity

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.

Low risk of inhibitor formation in haemophilia patients after a change in treatment from Chinese hamster ovary cell-produced to baby hamster kidney cell-produced recombinant factor VIII

2007 ◽  
Vol 98 (12) ◽  
pp. 1188-1192 ◽  
Author(s):  
Jude Smith ◽  
Mary Kavanagh ◽  
Beatrice Nolan ◽  
Barry White ◽  
Evelyn Singleton
Keyword(s):  
Factor Viii ◽  
Chinese Hamster Ovary ◽  
De Novo ◽  
Chinese Hamster ◽  
Low Risk ◽  
Recombinant Factor Viii ◽  
Phase Iii ◽  
Ovary Cell ◽  
Haemophilia A ◽  
Baby Hamster Kidney

SummaryThis retrospective survey of haemophilia A patients from multiple treatment centres in Ireland assessed the development of inhibitors following a switch in the prescribed treatment from recombinant factor VIII (rFVIII) produced by Chinese hamster ovary (CHO) cells (rFVIII-CHO) to rFVIII produced by baby hamster kidney (BHK) cells (rFVIII-BHK). Ninety-four patients participated in the survey. Most patients (89.4%) had severe haemophilia. One of 77 (1.3%) patients with no inhibitor history developed an inhibitor. This was a patient with moderate haemophilia A who developed a transient, low-titre (1 BU) de novo inhibitor following surgery. Recurrent inhibitors were detected in three of 17 patients with an inhibitor history during the 20-month post-switch study period. All patients continued on rFVIII-BHK therapy, and all tested negative for inhibitors at the time of their last inhibitor assay during the observation period. These results are consistent with the low levels of inhibitor formation demonstrated in phase III studies of previously treated patients receiving BHK-produced rFVIII and support the low risk of inhibitor formation following a change from rFVIIICHO to rFVIII-BHK.

scholarly journals Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes.

1990 ◽  
Vol 110 (3) ◽  
pp. 651-660 ◽  
Author(s):  
T Tsukamoto ◽  
S Yokota ◽  
Y Fujiki
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Ovary Cell ◽  
Wild Type ◽  
Normal Cells ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
In The Wild

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.

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